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At least 325 records · Page 18Linked to original sources
PubMed2002

Mutational analysis of the class I beta-tubulin gene in human breast cancer.

Non-small cell lung cancers have a high incidence of somatic mutations of the beta-tubulin (class I) gene, suggesting involvement in the acquisition of resistance to taxanes, which exert their effects through binding to beta-tubulin. Since taxanes are often used in the treatment of breast cancer, we carried out a mutational analysis of the class I beta-tubulin (GenBank accession AF070600) gene in breast cancer. We paid special attention to the primer design so as not to amplify the pseudogenes. We identified 1 somatic mutation, codon 306 [Arg (CGC) to Cys (TGC)], and 2 genetic polymorphisms, codon 217 [Leu (CTG) to Leu (CTA)] and (C to T) at 57 bases downstream from exon 4. Our results suggest that acquisition of resistance to taxanes is unlikely to be explained by somatic mutations of the class I beta-tubulin gene in most breast cancers. In addition, the overestimation of the incidence of somatic mutations of the class I beta-tubulin gene due to the pseudogenes is discussed.

Base Sequence↗
PubMed2003

Mutation analysis impact on the genetic counseling of sporadic hemophilia B families.

Previous studies have shown that hemophilia B (HB) is the result of several different mutations, mostly single nucleotide substitutions, in the factor IX (FIX) gene. In order to evaluate the impact of mutation analysis on genetic counseling in sporadic and uninformative HB familial pedigrees, we re-analyzed by the conformation sensitive gel electrophoresis (CSGE) technique 14 patients, previously studied by restriction fragment length polymorphisms (RFLPs). A single mutation was present within the FIX gene of each patient: 12 mutations were single base substitutions, 1 was a base insertion, and 1 was a four nucleotide deletion; 4/12 mutations have not been described so far. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied; 3/12 de novo events were found in maternal meioses with a 25% mutation rate. Identification of the genetic defect was also successfully applied to three prenatal diagnoses.

DNA Mutational Analysis↗
PubMed2001

Microsatellite instability and mutational analysis of transforming growth factor beta receptor type II gene (TGFBR2) in sporadic ovarian cancer.

AIMS: To investigate the possible role of mutations in the transforming growth factor beta receptor type II gene (TGFBRII) in ovarian cancer and its relation to microsatellite instability (MSI), 43 sporadic ovarian tumours were analysed for mutations over the entire coding region of the TGFBRII gene. METHODS: Mutational analysis was performed using the polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) gel analysis, and direct sequencing. MSI analysis included both mononucleotide and dinucleotide microsatellite markers used for radiolabelling and gene scanning. RESULTS: No pathogenic mutations were detected, although sequencing of the polyadenine (poly A) tract in exon 3 using conventional techniques revealed a spurious frameshift mutation that was not present in the same samples analysed using a proofreading Taq polymerase. MSI analysis demonstrated an MSI negative phenotype in 40 of the 43 tumours. None of the three MSI positive tumours demonstrated MSI for mononucleotide markers only. CONCLUSIONS: These findings suggest that: (1) MSI (both conventional and mononucleotide) is infrequent in ovarian cancer and (2) inactivation of the MSH2, MLH1, and MSH6 mismatch repair genes and TGFBR2 gene mutations do not play a major role in ovarian cancer tumorigenesis. The spurious TGFBR2 frameshift mutations detected by sequencing after conventional PCR underline the importance of confirming putative mutations in repetitive sequences by alternative methods.

Base Pair Mismatch↗
PubMed2006

SIMPLE mutation analysis in dominant demyelinating Charcot-Marie-Tooth disease: three novel mutations.

Charcot-Marie-Tooth disease type 1C (CMT1C) is caused by mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE). We analyzed the coding sequence of SIMPLE in DNA of 53 unrelated cases of dominant demyelinating CMT disease with no mutations in PMP22, GJB1, MPZ, EGR2, and NEFL genes. Four different missense mutations were observed in six families. The mutation Gly112Ser was found in two families confirming its frequent occurrence in SIMPLE mutations. Three novel mutations were also identified: Ala111Gly (two families), Pro135Ser, and Pro135Thr. Familial studies revealed that all carriers of mutations (n = 38), aged from 1 to 78 years, were symptomatic, notably children under 10 years (n = 8). Motor conduction velocities in the median nerve ranked from 16.4 to 32.8 m/s (n = 20). In our series of 968 unrelated dominant demyelinating CMT cases (1992-2005), the percentage of SIMPLE mutations was 0.6 (6/968).

Adolescent↗
PubMed1998

Mutational analysis of a satellite phage activator.

The late gene activator, Delta, of satellite phage P4 is more efficient than the Delta of satellite phage phiR73 in utilizing a P2 helper prophage that lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by the fact that this protein contains two tandem phiR73 Delta-like domains. We performed a mutational analysis of phiR73 Delta, in order to select mutations that might not be found using P4 Delta. The host RNA polymerase alpha subunit mutation rpoA155 (L289F) blocks the growth of P2, P4, and P4 carrying the delta gene of phiR73. A mutant of this latter phage that can grow in the presence of rpoA155 carries a V19M mutation in phiR73 Delta. This suggests that aa 19 contacts RNA polymerase, in addition to the aa residues 13, 42 and 44, that have been implicated in interactions with RNA polymerase by previous mutational analyses of P2 ogr and P4 delta. In corroboration of the proposed role of the regions at aa residues 19, 42, and 44, we found phiR73 Delta mutations in these regions that showed a reduced activation of late gene expression, but a normal ability to bind to late gene promoters. All activators in the Delta class contain four Cys residues that bind Zn2+. Mutation of these aa residues in phiR73 Delta eliminated late gene activation. Spectroscopic analysis of these mutant proteins revealed that they were unable to bind Zn2+. Histidine residues were substituted for two of the Cys residues (C30 and C35), changing a C2C2 type Zn-binding motif to a C2H2 motif. Although His residues are used in coordinating Zn2+ in other proteins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+.

Bacteriophages↗
PubMed1998

Presymptomatic testing for Huntington's disease by linkage and by direct mutation analysis: comparison of uptake of testing and characteristics of test applicants.

In Edinburgh, we have compared presymptomatic testing by linkage and by direct mutation analysis by investigating the demand for testing and characteristics of test applicants. Annual new requests for the direct test (DT) are now double the peak with the linkage test (LT) but only 6% individuals have requested re-testing. DT applicants were older with a smaller proportion having lived with an affected relative that LT applicants. This was because many were relatives of newly diagnosed first known cases in their family. This may also explain why DT applicants were less likely to expect a negative result and more likely to be uncertain about their risk. A greater proportion of DT applicants first heard about the test from relatives or their GP than LT applicants who were more likely to hear from Genetic Centre. The demand for follow-up by the Geneticist/Genetic Nurse was much less for DT than for LT applicants largely due to the support offered by the HD Advisors.

Adult↗
PubMed1999

Resequencing and mutational analysis using oligonucleotide microarrays.

Oligonucleotide microarray (DNA chip)-based hybridization analysis is a promising new technology which potentially allows rapid and cost-effective screens for all possible mutations and sequence variations in genomic DNA. Here, I review current strategies and uses for DNA chip-based resequencing and mutational analysis, the underlying principles of experimental designs, and future efforts to improve the sensitivity and specificity of chip-based assays.

Animals↗
PubMed2003

CpG island methylation status and mutation analysis of the RB1 gene essential promoter region and protein-binding pocket domain in nervous system tumours.

A series of 136 nervous system tumours were studied to determine the methylation status of the CpG island contained within the promoter region of the RB1 gene, as well as mutation analysis of the essential promoter region and exons 20-24 (and surrounding intronic regions) coding for the protein-binding pocket domain. Methylation of the RB1 CpG island was detected in 26 samples corresponding to nine glioblastomas, three anaplastic astrocytomas, one mixed oligo-astrocytoma, one ependymoma, two medulloblastomas, two primary central nervous system lymphomas, two neurofibrosarcomas, and six brain metastasis from solid tumours. No inactivating mutations were found within the RB1 promoter region, whereas one glioblastoma and one oligodendroglioma displayed similar sequence variations consisting of 12 and 8 base pair deletions at intron 21. These results suggest that RB1 CpG island hypermethylation is a common epigenetic event that is associated with the development of malignant nervous system tumours.

CpG Islands↗
PubMed1999

Genomic organization of the human galpha14 and Galphaq genes and mutation analysis in chorea-acanthocytosis (CHAC).

Chorea-acanthocytosis (CHAC) (OMIM 200150) is a rare neurological syndrome characterized by neurodegeneration in combination with morphologically abnormal red cells (acanthocytosis). A partial yeast artificial chromosome contig of the CHAC critical region on chromosome 9q21 has been constructed, and 21 expressed sequence tags have been mapped. We have subsequently cloned Galpha14, a member of the G-protein alpha-subunit multigene family, and have identified Galphaq in the contig. The genomic structure of both genes has been established after construction of a bacterial artificial chromosome contig that showed Galphaq and Galpha14 to be in a head-to-tail arrangement (Cen-Galphaq-Galpha14-qter). Northern analysis found Galphaq to be ubiquitously expressed and Galpha14 to display a more restricted pattern of expression. Mutation analysis of the coding regions and splice sites for Galphaq and Galpha14 in 10 affected individuals from different families identified no changes likely to cause disease; however, two distinct single nucleotide polymorphisms in the coding region of Galpha14 have been identified. This study has excluded two plausible candidate genes from involvement in CHAC and has provided a solid platform for a positional cloning initiative.

Acanthocytes↗
PubMed2006

Mutational analysis in 119 families with nephronophthisis.

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease (ESRD) in the first three decades of life. Six genes, NPHP1-6, have been reported, which when mutated result in NPHP. Our aim was to examine 119 families with NPHP and absence of homozygous NPHP1 deletions for mutations in NPHP2-6 and the two candidate genes BCL2 and CYS1. The 119 individuals affected with NPHP were selected from unrelated families, in which homozygous NPHP1 deletions were excluded. A combination of CEL-1 endonuclease digestion and direct sequencing was used for focused mutational analysis in this cohort. All individuals were examined for homozygous deletions in NPHP1 and directly sequenced for BCL2 and CYS1. As selected by appropriate phenotype, 9%, 38%, 97%, 20% and 20% of individuals were examined for mutations in NPHP2, 3, 4, 5, and 6 respectively. No mutations in known NPHP genes or in the candidate genes, BCL2 and CYS1, were found sufficient to explain NPHP in affected individuals. These findings demonstrate the need to evaluate additional candidate genes as the cause of NPHP.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Cx26 deafness: mutation analysis and clinical variability.

Mutations in the gap junction protein connexin 26 (Cx26) gene (GJB2) seem to account for many cases of congenital sensorineural hearing impairment, the reported prevalence being 34-50% in autosomal recessive cases and 10-37% in sporadic cases. The hearing impairment in these patients has been described as severe or profound. We have studied 53 unrelated subjects with congenital non-syndromic sensorineural hearing impairment in order to evaluate the prevalence and type of Cx26 mutations and establish better genotype-phenotype correlation. Mutations in the Cx26 gene were found in 53% of the subjects tested, 35.3% of the autosomal recessive and 60% of the sporadic cases in our series. Three new mutations were identified. The hearing deficit varied from mild to profound even in 35delG homozygotes within the same family. No evidence of progression of the impairment was found. Alterations of the Cx26 gene account for a large proportion of cases of congenital non-syndromic sensorineural deafness, so it seems appropriate to extend the molecular analysis even to subjects with mild or moderate prelingual hearing impairment of unknown cause.

Adolescent↗
PubMed2004

RNA-editing terminal uridylyl transferase 1: identification of functional domains by mutational analysis.

The catalytic, RNA-binding and oligomerization domains of the RNA-editing terminal uridylyl transferase 1 (RET1) from Leishmania tarentolae mitochondria were characterized by mutational analysis. Significant N- and C-terminal portions of the protein were found to be dispensable for UTP polymerization in vitro. Changes of conserved amino acids in the active site demonstrated a general similarity of sugar-phosphate moiety recognition of the incoming ribonucleotide triphosphate by RET1 and eukaryotic poly(A) polymerases. Overlapping RNA-binding and oligomerization regions were mapped to the C-terminal region, which is conserved only among trypanosomatid RET1 enzymes. In the absence of an RNA primer, RET1 can use UTP itself to initiate nucleotide transfer and produce poly(U) molecules of several hundred nucleotides. An N-terminal zinc finger motif is essential for enzyme activity; deletion of this motif or chelation of zinc inhibits activity.

Amino Acid Motifs↗
PubMed1997

An efficient and reliable multiplex PCR-SSCP mutation analysis test applied to the human E-cadherin gene.

The invasion suppressor gene E-CADHERIN (CDH1) is downregulated in a large variety of human carcinomas. Up to now, mutational analysis of the CDH1 gene has been described for 325 tumors derived from only four different tissue types. A simple but sensitive mutation detection assay is needed to screen many more tumor types, possibly bearing E-cadherin inactivating mutations. For that purpose, we developed a multiplex PCR-SSCP analysis for all 16 CDH1 exons. Ease of experimentation was combined with reliable sensitivity. Indeed, the present multiplex analysis reduces the number of manipulations to 50%, while the mutation detection turned out to be highly efficient and sensitive.

Base Sequence↗
PubMed1994

Structural inferences of the ETS1 DNA-binding domain determined by mutational analysis.

The ets family of transcription factors is characterized by a conserved region that harbors the DNA-binding activity. We performed extensive deletion and mutational analyses, as well as DNA-peptide interaction studies necessary to identify the determinants of the DNA-binding activity of the ETS1 oncoprotein. We found that amino acids beyond the 85 amino acid conserved region are required in order to afford maximum DNA-binding activity in a heterologous system. Mutation throughout the binding domain can have a detrimental effect on binding activity, indicating that proper folding of the entire domain is necessary for DNA binding. A peptide, as small as 37 residues (K37N), derived from the basic region of the ETS1 binding domain, is sufficient to exhibit sequence-specific DNA recognition. Total randomization of Lysine 379, Lysine 381 and Arginine 391 within this region fails to provide functional substitutions, indicating that these specific amino acids within the basic region are required for binding. Transactivation activity of the ETS1 proteins bearing mutations was consistent with their DNA-binding activity, indicating that the primary (if not only) function of this domain is to provide sequence-specific DNA recognition activity. Our mutational analysis, as well as modeling predictions, lead us to propose a helix-turn-helix structure for the basic region of the ETS1 binding domain that is able to interact directly with DNA. We also propose that the hydrophobic alpha-helical region, surrounding tryptophan 338, is fundamental for proper protein folding and functioning of the ets binding domain.

Amino Acid Sequence↗
PubMed1999

Jagged-1 mutation analysis in Italian Alagille syndrome patients.

Alagille syndrome (AGS) is an autosomal dominant disorder with developmental abnormalities affecting the liver, heart, eyes, vertebrae, and craniofacial region. The Jagged-1 (JAG1) gene, which encodes a ligand of Notch, has recently been found mutated in AGS. In this study, mutation analysis of the JAG1 gene performed on 20 Italian AGS patients led to the identification of 15 different JAG1 mutations, including a large deletion of the 20p12 region, six frameshift, three nonsense, three splice-site, and two missense mutations. The two novel missense mutations were clustered in the 5' region, while the remaining mutations were scattered throughout the gene. The spectrum of mutations in Italian patients was similar to that previously reported. We also studied in detail a complex splice site mutation, 3332dupl8bp, which was shown to lead to an abnormal JAG1 mRNA, resulting in a premature stop codon. With the exception of the missense mutations, the majority of the JAG1 mutations are therefore likely to produce truncated proteins. Since the phenotype of the patient with a complete deletion of the JAG1 gene is indistinguishable from that of patients with intragenic mutations, our study further supports the hypothesis that haploinsufficiency is the most common mechanism involved in AGS pathogenesis. Furthermore, our data confirmed the absence of a correlation between the genotype of the JAG1 gene and the AGS phenotype.

Alagille Syndrome↗
PubMed1994

Mutational analysis of the first 14 exons of the adenomatous polyposis coli (APC) gene.

In the present study, the polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) technique has been applied to the mutation analysis of the adenomatous polyposis coli (APC) gene. We examined the first 14 exons of the APC gene in 46 polyposis coli patients. Five germline mutations were observed, including a single-nucleotide substitution and small (1-4 bp) deletions leading, in 4 cases, to a stop codon. A missense mutation in exon 3 and a 1 bp deletion in exon 4 of the APC gene were observed in patients presenting with the attenuated form of FAP.

Adenomatous Polyposis Coli↗
PubMed1993

Mutational analysis of the regulatory region of the srfA operon in Bacillus subtilis.

Transcription of the Bacillus subtilis srfA operon is dependent on the transcriptional activator ComA. Mutational analysis of the srfA regulatory region suggests that two regions of dyad symmetry upstream of the srfA promoter may function in transcriptional activation by facilitating a cooperative interaction between ComA dimers.

Bacillus subtilis↗
PubMed1995

Diagnosis of acute intermittent porphyria in northern Sweden: an evaluation of mutation analysis and biochemical methods.

OBJECTIVE: To validate the use of a recently observed guanine to adenine mutation in exon 10 in the porphobilinogen deaminase (PBGD) gene as a diagnostic marker of acute intermittent porphyria (AIP). To evaluate the efficiency of the traditional biochemical diagnostic methods. DESIGN: Matched and blinded case-control study (1:4). SETTING: A primary health care centre in Arjeplog, the National Porphyria Research Unit and a department of clinical genetics in Stockholm. SUBJECTS: A total of 48/49 (98%) patients over the age of 15 years living in Arjeplog with AIP, diagnosed according to standard clinical and biochemical criteria. For each AIP patient, four controls were matched for age, sex and geographical area and 164/196 (86%) participated. In the validity study, 35 patients were selected as indisputable AIP gene carriers, according to strict biochemical criteria, and 92 matched controls were selected with strict exclusion criteria. MAIN OUTCOME MEASURES: Validity, specificity and sensitivity of DNA diagnosis for this AIP mutation. Specificity and sensitivity of traditional biochemical methods. RESULTS: Validity study: the mutation was found in all 35 individuals classified as carriers of AIP. None of the 92 controls had the mutation. Evaluation study: all 48 AIP gene carriers, diagnosed by traditional methods, had the mutation, as had one of the control persons. In an inconclusive group of five persons with heredity for AIP, two had a positive DNA test. CONCLUSIONS: The PBGD mutation analysis was found to have full specificity and sensitivity and can be used as the sole diagnostic method in the family complex studied, representing the major AIP mutation in Sweden. The traditional diagnostic methods, used in optimal combinations, work in most cases, but they do not show high precision. However, they must be used when the specific mutation in the PBGD gene is not known.

Adolescent↗