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PubMed2003

Mutational analysis of Noxa gene in human cancers.

There has been mounting evidence that dysregulation of apoptosis is involved in the mechanisms of cancer development and somatic mutations of apoptosis-related genes have been reported in human cancers. Noxa, a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family, is known to interact with anti-apoptotic Bcl-2 family members and induces apoptosis. The aim of this study was to explore the possibility that the Noxa gene is mutated in human cancers. We have analyzed the entire coding region and all splice sites of the Noxa gene for the detection of somatic mutations in a series of human cancers, including carcinomas from stomach, colon, liver, urinary bladder and lung by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), and DNA sequencing. We found one somatic mutation of the Noxa gene in a transitional cell carcinoma (TCC) of the urinary bladder. To evaluate the functional alterations of the mutant in apoptosis, we overexpressed the mutant and wild-type Noxa in 293T and HeLa cells, but could not find any significant difference in cell death between the wild-type and mutant Noxa. These data suggest that Noxa is rarely mutated in human carcinomas and that the contribution of Noxa gene mutation in the pathogenesis of human cancer might not be related to cell death mechanisms.

Adenocarcinoma↗
PubMed1999

Mutation analysis of the EMX2 gene in Kallmann's syndrome.

OBJECTIVE: To investigate the possibility that a mutation in the human EMX2 gene may be involved in Kallmann's syndrome. DESIGN: In vitro experiment. SETTING: Academic Medical Center. PATIENTS: One hundred and twenty patients with Kallman's syndrome or idiopathic hypogonadotrophic hypogonadism (IHH). INTERVENTION: Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOMES MEASURES: Single-stranded conformational polymorphism (SSCP) analysis was used to identify possible mutations of the EMX2 gene. RESULTS: One hundred and twenty patients with Kallmann's syndrome or IHH, had no mutations noted in this gene. CONCLUSION: It is unlikely that EMX2 mutations are a clinically significant cause of IHH or Kallman's syndrome.

Adolescent↗
PubMed1996

Mutation analysis of the BRCA1 gene in 23 families with cases of cancer of the breast, ovary, and multiple other sites.

Germline mutations in the BRCA1 tumour suppressor gene on chromosome 17q21 are responsible for approximately half of the cases of hereditary breast cancer, including the majority of familial breast/ovarian cancers. To increase our knowledge of the spectrum of BRCA1 mutations, we have extended our analysis to include patients with varied family histories of cancer of the breast, ovary, and at multiple other sites. We have analysed 23 unrelated familial cases using direct sequencing or a combination of dideoxy fingerprinting and sequencing procedures. Twenty one of these families contained three or more cases of breast or ovarian cancer and two families had one case of breast cancer diagnosed before the age of 40 and one case of ovarian cancer. The common frameshift mutation 5382insC was detected in two patients, and the 185delAG mutation was found in a family of Ashkenazi Jewish descent. The novel frameshift mutation 3450del4 (CAAG) was detected in a patient who developed breast cancer at the age of 28 and ovarian cancer at the age of 34. Three other women in this family were diagnosed with breast cancer at the ages of 26, 29, and 40. The novel framshift mutation 2953del3+C was found in a French Canadian woman who had developed two primary cancers of the breast at the age of 37 and 38 and renal cancer at the age of 38.

Adult↗
PubMed1997

Mutation analysis in 46 British and Irish patients with Gaucher's disease.

DNA from 46 unrelated patients with Gaucher's disease was analysed for 10 known mutations: 84GG(c84 G 85ins), N370S (c1226G), L444P (c1448C), R463C (c1504T), R496H (c1604A), IVS2+1, D409H (c1342C), RecNcil (c1448C-1498C), RecTL (c1342C-1498C), and c1263del (c1264-1318del). Fifty four mutations (90%) were identified in 30 patients with type I disease. These included a previously undescribed recombinant mutation RecA456P (c1448C-1484C). Thirteen (54%) of 24 type II alleles were identified, including one new point mutation N462K (c1503G) and one new 55bp deletion also incorporating the RecTL mutations c1263del+RecTL (c1264del-1498C). All four type III patients were homozygous for the L444P point mutation. Generally, patients with one copy of the N370S mutation had mild adult onset disease, regardless of the nature of their second mutation. Three exceptions had childhood onset disease and genotypes N370S/R463C, N370S/RecA456P, and N370S/?. The L444P/L444P genotype was thought to be associated with neurological disease. Two type I patients with this genotype who exhibited no central nervous system disease were identified, however. The R463C and c1263del mutations were found to be present at a higher frequency than reported in other populations and they should be included in any mutation screen of this population. The recombinant mutations RecA456P and c1263del+RecTL have not been previously described and are the fourth and fifth recombinant mutations identified in the glucocerebrosidase gene.

Adult↗
PubMed1996

Mutation analysis in 24 French patients with glycogen storage disease type 1a.

Both alleles of 24 French glycogen storage disease type 1a patients were sequenced: 14 different mutations allowed the identification of complete genotypes for all the patients. Nine new gene alterations are reported. Five mutations, Q347X, R83C, D38V, G188R, and 158 del C, account for 75% of the mutated alleles. These data show that the molecular pathology of the glucose-6-phosphatase gene is heterogeneous in this population. Complete genotyping of the index case by systematic sequencing is necessary to allow prenatal diagnosis in chorionic villi for at risk couples.

Base Sequence↗
PubMed1999

Mutational analysis of the PMS2 gene in sporadic endometrial cancers with microsatellite instability.

OBJECTIVE: Approximately 20% of endometrial tumors have a defect in DNA mismatch repair and exhibit microsatellite instability (MSI). We assessed the role of the PMS2 DNA mismatch repair gene in MSI-positive sporadic endometrial tumors. METHODS: We examined 40 sporadic endometrial tumor specimens with MSI. All 15 exons of the PMS2 gene were investigated for sequence alterations by single-strand conformational variant analysis. RESULTS: Twelve polymorphisms were identified, 8 of which were in the coding sequence. Four specimens revealed mutations in intronic sequences that are not predicted to affect the PMS2 mRNA. No mutations were detected within the coding region of the PMS2 gene. CONCLUSION: We conclude that structural mutations in the PMS2 gene are not responsible for defective DNA mismatch repair in sporadic endometrial cancers with MSI. The identification of single nucleotide polymorphisms in the PMS2 locus may aid in the mapping and characterization of genetic diseases.

Adenosine Triphosphatases↗
PubMed2004

Mutational analysis of the HIV-1 LTR as a promoter of negative sense transcription.

The HIV-1 gene promoter is a bi-directional promoter of transcription. We report the characterization of the negative sense promoter (NSP) by analysis of the effect on negative sense transcription of a series of LTR U3 region substitution mutants. Mutations in the region nt -58 to -183 (positive sense transcription initiation nt +1) reduced transcription to <15% of wild type NSP activity. This region, essential for NSP activity, was designated the core basal promoter. Over expression of NF-kappaB RelA(p65) and LEF-1 increased negative sense expression, as did over expression of H-ras oncogene, consistent with the presence of cognate sequence motifs for NF-kappaB, LEF-1 and RBF. We were also able to confirm that the NSP is a TATA-less promoter inhibited by HIV-1 Tat. Based on our findings, we propose a model for the interaction between the NSP and PSP, and the role of Tat in regulating the interaction.

DNA Mutational Analysis↗
PubMed2006

A mutational analysis of Caenorhabditis elegans in space.

The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.

Animals↗
PubMed2004

Mutational analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth neuropathy patients.

We examined CMT1A duplication of 17p11.2-p12, mutations of PMP22, MPZ (P0), GJB1 (Cx32), EGR2 and NEFL genes in 57 Korean families with patients diagnosed as having Charcot-Marie-Tooth (CMT) disease. The CMT1A duplication was present in 53.6% of 28 CMT type 1 patients. In the 42 CMT families without CMT1A duplication, 10 pathogenic mutations were found in 9 families. The 10 mutations were not detected in 105 healthy controls. Seven mutations (c.318delT (p.Ala106fs) in PMP22, c.352G>A (p.Asp118Asn), c.449-1G>T (3'-splice site), c.706A>G (p.Lys236Glu) in MPZ, c.407T>C (p.Val136Ala)[corrected], c.502T>C (p.Cys168Arg) in GJB1, and c.1001T>C (p.Leu334Pro) in NEFL) were determined to be novel. The mutation frequencies of PMP22 and MPZ were similar to those found in several European populations, however, it appeared that mutations in GJB1 are less frequent in East Asian CMT patients than in Eur opean patients. We described the identified mutations and phenotype-genotype correlations based on nerve conduction studies.

Charcot-Marie-Tooth Disease↗
PubMed1993

Mutational analysis of the interaction between ecdysteroid receptor and its response element.

The interaction between the partially purified ecdysteroid receptor (EcR) and the mutated ecdysteroid-response element (EcRE) from the hsp27 gene promoter was studied using the gel retardation competition assay. The results suggest that the EcR-hsp27 EcRE contact sites are made predominantly by base pairs which are at positions -7, -6, -5, -2, -1 and +2, +5, +6 of the hsp27 EcRE palindrome. An increase or decrease in the spacing between the half-palindromes reduces the affinity of the hsp27 EcRE to the receptor, while a mutation of the central A/T base pair to C/G has practically no effect on EcR binding. Unlike the glucocorticoid-response element and the estrogen-response element, the base pairs placed at positions -3, -4 and +1, +3, +4 of the hsp27 EcRE palindrome can be mutated without effect on the EcR binding.

Animals↗
PubMed1998

Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe-2S] cluster of the cytochrome bc1 complex in Paracoccus denitrificans.

Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster. In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567-579]. They are conserved in all known Rieske iron-sulfur proteins from bc1 complexes including that from P. denitrificans. Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain. The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.

Amino Acid Sequence↗
PubMed2001

Cloning and mutation analysis of the human potassium channel KCNQ2 gene promoter.

Benign familial neonatal convulsions (BFNC) have been previously found to be associated with mutations within the coding region of KCNQ2. We have now cloned and analyzed the promoter region of the human KCNQ2 gene. 5'-RACE identified a transcription start site (TSS) located 200 bp upstream of the ATG start codon. The TSS is located close to a repetitive region containing seven copies of a degenerate 42-mer repeat. Several different luciferase (LUC) reporter plas- mids containing fragments from the KCNQ2 5'-flanking region were constructed and expressed in NT2N and SH-SY5Y cell lines. A core promoter region was found to be located between bp 20 and bp 74 upstream of the TSS. Neither the promoter region nor the repetitive region showed any mutations in 13 index patients from unrelated BFNC families.

5' Flanking Region↗
PubMedDate not supplied

Germline mutational analysis of presenilin 1 and APP genes in Jewish-Israeli individuals with familial or early-onset Alzheimer disease using denaturing gradient gel electrophoresis (DGGE).

Germ line mutations in three genes have been detected in patients with familial Alzheimer's disease (FAD) and sporadic, early onset disease: amyloid precursor protein (APP), presenilin 1 (PS-1), and presenilin 2 (PS-2). The relative proportions in which mutations in these genes occur among AD patients in Israel has not been evaluated. To that end, we screened 52 Jewish-Israeli patients with AD: 22 with sporadic, early-onset disease (below 65 years), and 30 with FAD. Mutation screen employed denaturing gradient gel electrophoresis (DGGE) of exon-specific PCRs and restriction enzyme digest. Five patients from three different families displayed mutations within the PS-1 gene: three patients of one family showed a mis-sense mutation in codon 120 (Glu 120Lys), and two other unrelated patients showed an identical mis-sense mutation in codon 318 (Glu318Gly). No patient showed an abnormal migration on DGGE (for APP) or mutant restriction digest pattern (for PS-2) genes. These data may indicate the existence of another familial Alzheimer disease (FAD) gene locus in the Israeli Jewish population.

Adult↗
PubMed1997

Mutation analysis of BRCA1 and BRCA2 in a male breast cancer population.

A population-based series of 54 male breast cancer cases from Southern California were analyzed for germ-line mutations in the inherited breast/ovarian cancer genes, BRCA1 and BRCA2. Nine (17%) of the patients had a family history of breast and/or ovarian cancer in at least one first-degree relative. A further seven (13%) of the patients reported breast/ovarian cancer in at least one second-degree relative and in no first-degree relatives. No germ-line BRCA1 mutations were found. Two male breast cancer patients (4% of the total) were found to carry novel truncating mutations in the BRCA2 gene. Only one of the two male breast cancer patients carrying a BRCA2 mutation had a family history of cancer, with one case of ovarian cancer in a first-degree relative. The remaining eight cases (89%) of male breast cancer with a family history of breast/ovarian cancer in first-degree relatives remain unaccounted for by mutations in either the BRCA1 gene or the BRCA2 gene.

Aged↗
PubMed1996

RET protooncogene mutational analysis in multiple endocrine neoplasia syndrome type 2B: case report and review of the literature.

Multiple endocrine neoplasia, type 2B (MEN 2B), is a phenotypic variant of a group of autosomal-dominant neurocristopathies. MEN 2B is associated with medullary thyroid carcinoma and pheochromocytoma with oral, ocular, and alimentary submucosal ganglioneuromas and Marfanoid body features. Approximately 50% of cases are thought to be spontaneous mutations. The RET protooncogene (RET) is a 21-exon gene encoding a tyrosine kinase receptor. A codon 918 germ line mutation, which converts a highly conserved methionine to a threonine in the intracellular tyrosine kinase portion of this receptor of RET, has been identified in 95% of patients with MEN 2B. This mutation is easily detected by a direct deoxyribonucleic acid sequencing or restriction enzyme (Fok 1) analysis of amplified polymerase chain reaction products. The RET gene is normally expressed in the oral and gastrointestinal submucosal neural ganglia, and the codon 918 mutation is thought to cause neuromas by virtue of its transforming activity in these ganglia. Identifying clinical features of MEN 2B in an 11-year-old boy by an oral pathologist led to confirmation by mutational analysis. Before genetic testing was available, the patient, and at a later date his mother, underwent thyroidectomies based solely on biochemical testing. Results indicated the patient had the codon 918 mutation, whereas his phenotypically normal mother, father, and older brother had normal RET analyses. Studies in families have demonstrated that the mutant allele is derived from the father with possible acquisition during spermatogenesis. We believe the mother of our affected patient to be normal; the absence of phenotypic features of MEN 2B and a normal genotype suggest her calcitonin abnormalities and minimal evidence for C-cell hyperplasia were inconsequential. Molecular analysis for RET abnormalities will likely supplant biochemical methods of diagnosis in patients with MEN 2B.

Carcinoma, Medullary↗
PubMed2006

p53 Mutation analysis in breast tumors by a DNA microarray method.

The p53 gene acts as a regulator of cell growth and DNA repair in normal cells; inactivation of the gene seems to lead to cancer. It is the most commonly mutated gene in human cancers, and a high-throughput sequencing method is needed for cancer etiology studies using large sample sets. In our population-based case-control study of breast cancer, the p53 gene was amplified by PCR for 392 subjects from seven hospitals in Western New York using the Affymetrix GeneChip technology. One hundred thirty-eight (35%) of the breast tumors had p53 mutations, of which 88% were located in exons 5 to 8. New hotspots were identified at codons 179, 195, 196, 213, 217, 249, 254, 278, 281, and 298, and previously reported hotspots were found at codons 175, 248, and 273. Manual sequencing for exons 5 to 9 of the p53 gene was done for 139 tumors to validate the Affymetrix assay. The two methods had 100% concordance for mutations detectable by the Affymetrix assay. We also successfully assayed paraffin-embedded breast and lung tumors from as early as 1958 and employed a nested PCR strategy to improve weak PCR amplification. To have statistical power, the investigation of gene environment interactions and cancer requires a large number of tumor analyses, which are frequently only available from archived tissue from multiple sources. We have shown the utility of the Affymetrix GeneChip method under these challenging conditions and provided new data for the mutational spectra of breast cancer in a population-based study.

Breast Neoplasms↗