Biomedical subjects

Kathleen M Murphy

Publications and source records attributed to Kathleen M Murphy.

30 records · Page 2Linked to original sources

PubMed2004

Identification of a functional mutation in pp32r1 (ANP32C).

No mutations or polymorphisms have previously been reported in pp32r1 (ANP32C; GenBank: AF008216.1). pp32r1 is part of the highly conserved ANP32 family, some of whose members are associated with control of histone acetylation, mRNA stability, and specialized forms of apoptosis. Although 87.6% identical at the protein level, pp32r1 is functionally distinct from pp32 (ANP32A) in its failure to suppress oncogenesis in in vitro transformation systems and its tumorigenicity in in vivo assays. The present study found that pp32r1 expression levels vary among human tumor cell lines, with the highest levels found in prostatic adenocarcinoma cell lines. pp32r1 also appears to be polymorphic at nucleotide g.4520 and nucleotide g.4664 in human tobacco-associated oral mucosal lesions, human fibroblast cell lines, and several carcinoma cell lines. PC-3 human prostatic adenocarcinoma cells likewise appear to be polymorphic at these loci, but additionally contain a g.4870T>C transversion mutation. The mutation results in a p.Tyr140His substitution, which lies in a functionally important region of the molecule. In the PC-3 prostate cancer line, the mutation is either homozygous, or hemizygous accompanied by loss of heterozygosity. ACHN cells stably transfected with pp32r1 containing this mutation showed a markedly increased rate of growth. The pp32r1 mutation could thus be causally associated with the neoplastic growth properties of PC-3, and be of potential clinical significance.

Amino Acid Sequence↗

PubMed2004

Molecular diagnosis of metastasizing oligodendroglioma: a case report.

We report the case of a suspicious parotid mass in which molecular determination of loss of heterozygosity (LOH) of chromosome arms 1p and 19q in combination with cytologic and immunohistochemical analysis defined the tumor to be metastatic oligodendroglioma. The patient was a 41-year-old woman who developed a World Health Organization grade II oligodendroglioma in her right frontal lobe at age 32, for which no adjuvant chemo- or radiotherapy was administered. Five years following this diagnosis, radiological assessment revealed a 10-centimeter mass in the tumor bed, suspicious for a recurrence. Resection of this lesion revealed an anaplastic oligodendroglioma (grade III) and adjuvant radiotherapy was given. Eleven months after this surgery the patient presented with a 5.5-cm subcutaneous, non-mobile, non-tender mass in the region of the right parotid gland. Fine needle aspiration (FNA) yielded highly cellular material, morphologically and immunohistochemically suspicious for oligodendroglioma. Molecular analysis of microsatellite loci residing on chromosome arms 1p and 19q was performed using DNA extracted from the patient's recurrent brain oligodendroglioma and the FNA specimen. This analysis revealed evidence of LOH at all eight of the microsatellite loci tested. The combination of cytologic and molecular findings defined the extracranial tumor to be metastatic oligodendroglioma.

Adult↗

PubMed2004

FLT3 mutations in myeloid sarcoma.

Myeloid sarcoma is an extramedullary tumour that typically occurs in the setting of acute myeloid leukaemia (AML), or myeloproliferative disorders. In AML, two types of mutations in Fms-like tyrosine kinase 3 (FLT3) have been described; internal tandem duplications (ITD) and point mutations at aspartic acid residue 835 (D835). We analysed 24 myeloid sarcoma specimens from 20 patients for FLT3 ITD and D835 mutations. FLT3 ITD mutations were identified in three of 20 cases (15%); no D835 mutations were identified. The ITD inserts ranged in size from 33 to 198 base pairs (bp) and represented approximately 20-40% of the FLT3 alleles. Two cases showed discordance in FLT3 ITD mutational status. In one case, the leukaemia specimen was positive for a FLT3 ITD mutation and the myeloid sarcoma specimen was negative. In the second case, the myeloid sarcoma was positive for a FLT3 ITD mutation at diagnosis, but negative in subsequent relapse samples. Our findings suggest that small molecule inhibitors of FLT3 may be useful therapeutic agents for treatment of myeloid sarcomas-containing FLT3 mutations, however, the potential for discordance between the leukaemia and myeloid sarcoma, necessitates that the myeloid sarcoma tumour itself be analysed for FLT3 mutations.

Acute Disease↗

PubMed2003

Evidence of selection for clones having genetic inactivation of the activin A type II receptor (ACVR2) gene in gastrointestinal cancers.

The activin signaling pathway parallels the transforming growth factor (TGF)-beta pathway. Both use extracellular ligands and cell surface receptors that are structurally and functionally related, as well as the same intracellular mediators (SMADs 2-4) to transmit these signals. Members of both pathways have been characterized previously as tumor suppressor genes on the demonstration of inactivating mutations in human neoplasms, e.g., genetic inactivation of the activin type I receptor was reported recently in pancreatic cancer. Here, we present evidence of selection for mutations of the activin A type II receptor (ACVR2) gene during human gastrointestinal carcinogenesis. Two 8-bp polyadenine tracts of the ACVR2 gene are targets for inactivating frameshift mutations in gastrointestinal neoplasms having microsatellite instability (MSI). These mutations are similar to those of the 10-bp polyadenine tract within the TGF-beta type II receptor (TGFBR2), a well-characterized target of frameshift mutations in the same neoplasms. We identified biallelic mutations of ACVR2 in 25 of 28 MSI colorectal and pancreatic cancers. In addition, a mutation in the ACVR2 gene combined with loss of the wild-type allele was found in a non-MSI pancreatic cancer. This evidence is compatible with a high degree of selection for inactivation of the ACVR2 gene in tumorigenesis, supporting ACVR2 as a candidate tumor suppressor gene in gastrointestinal cancers.

Activin Receptors, Type II↗

PubMed2003

Detection of FLT3 internal tandem duplication and D835 mutations by a multiplex polymerase chain reaction and capillary electrophoresis assay.

FLT3 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells and plays an important role in stem cell survival and differentiation. Two different types of functionally important FLT3 mutations have been identified. Internal tandem duplication mutations arise from duplications of the juxtamembrane portion of the gene and result in constitutive activation of the FLT3 protein. This alteration has been identified in approximately 20% to 30% of patients with acute myelogenous leukemia and appears to be associated with a worse prognosis. The second type of FLT3 mutation, missense mutations at aspartic acid residue 835, occurs in approximately 7.0% of acute myelogenous leukemia cases. These mutations also appear to be activating and to portend a worse prognosis. Identification of FLT3 mutations is important because it provides prognostic information and may play a pivotal role in determining appropriate treatment options. We have developed an assay to identify both internal tandem duplication and D835 FLT3 mutations in a single multiplex polymerase chain reaction. After amplification, the polymerase chain reaction products are analyzed by capillary electrophoresis for length mutations and resistance to EcoRV digestion. Here we describe the performance characteristics of the assay, assay validation, and our clinical experience using this assay to analyze 147 clinical specimens.

Alleles↗

PubMed2003

A single nucleotide primer extension assay to detect the APC I1307K gene variant.

Adenomatous polyposis coli (APC) is a tumor suppressor gene important in colorectal tumorigenesis. A genetic variant of APC, I1307K, results from a T-to-A transversion at nucleotide 3920 which converts the wild-type sequence to a homopolymer tract (A(8)). The I1307K alteration is not itself oncogenic, but creates a hypermutable region (A(8)) that is prone to frame-shift mutations. The APC I1307K variant occurs in approximately 6% of the Ashkenazi Jewish population and is reported to approximately double an individual's risk for colorectal cancer. Here we describe a single nucleotide primer extension assay for the detection of the APC I1307K mutation. Following PCR amplification, nucleotide 3920 of the APC gene is directly sequenced using single nucleotide primer extension technology. The assay is in a multiplex format allowing simultaneous forward and reverse sequencing of the I1307K variant, which provides an internal, independent confirmation of each testing result. The assay was validated against 60 samples previously characterized by an allele-specific oligonucleotide (ASO) hybridization assay, with 100% concordance of results. Compared to the ASO assay, this single nucleotide primer extension assay requires significantly less technical time to perform, and has a greatly increased throughput capacity. The single nucleotide extension assay provides a highly sensitive and specific assay to identify individuals with the APC I1307K gene variant who may benefit from increased colorectal screening.

Base Sequence↗

PubMed2003

Molecular diagnosis of oligodendroglioma in paraffin sections.

Distinction of oligodendrogliomas from other gliomas is clinically important, but the histologic diagnosis of oligodendroglioma has been a difficult and notoriously subjective task. Testing for loss of heterozygosity (LOH) on chromosomal arms 1p and 19q, the genetic signature of oligodendroglioma, has emerged as a useful, objective adjunct to the traditional histologic evaluation. However, LOH testing of glioma specimens has not yet been widely implemented, presumably because of a lack of a practical LOH assay. We describe a 1p and 19q LOH assay suitable for routine diagnostics. In contrast to traditional microsatellite-based LOH analysis, we show that detection of LOH is usually possible even without normal tissue or blood from the same patient. A small area of tumor on a single paraffin section is sufficient for the assay. The assay protocol consists of a one-step DNA extraction, multiplex PCR for microsatellites on 1p and 19q, and capillary electrophoresis of the PCR products. LOH is detected by analysis of the allelic patterns and by integration of data from multiple highly polymorphic microsatellites. In a validation study on 19 gliomas, the results were concordant with results obtained by established methods and correlated well with histologic diagnoses. Because only a paraffin section is required, the pathologist can perform both the traditional histopathologic evaluation and this supporting molecular assay from the material at hand.

Brain Neoplasms↗

PubMed2003

Mutation and single nucleotide polymorphism detection using temperature gradient capillary electrophoresis.

Rapid, high-throughput mutation and single nucleotide polymorphism detection technologies are necessary to identify sequence alterations responsible for human disease. Several screening techniques have been developed as alternatives to the costly and time-consuming task of direct gene sequencing. Unfortunately, many of these techniques have relatively low mutation detection sensitivities and/or require significant up-front assay optimization. Temperature gradient capillary electrophoresis is a relatively new mutation screening technology that capitalizes on the denaturing effects of temperature and the high resolution capacity of capillary electrophoresis to detect heteroduplexes formed between mutant and wild type gene sequences. The utility of temperature gradient capillary electrophoresis for the detection of known sequence alterations and as a tool for mutation discovery is reviewed.

DNA Mutational Analysis↗

PubMed2002

Evaluation of candidate genes MAP2K4, MADH4, ACVR1B, and BRCA2 in familial pancreatic cancer: deleterious BRCA2 mutations in 17%.

It is estimated that familial aggregation and genetic susceptibility play a role in as many as 10% of pancreatic ductal adenocarcinomas. To investigate the role of germ-line mutations in the etiology of pancreatic cancer, we have analyzed samples from patients with pancreatic cancer enrolled in the NFPTR for mutations in four tumor suppressor candidate genes: (a) MAP2K4; (b) MADH4; (c) ACVR1B; and (d) BRCA2 by direct sequencing of constitutional DNA. These genes are mutated in clinically sporadic pancreatic cancer, but germ-line mutations are either not reported or anecdotal in familial pancreatic cancer. Pancreatic cancer patient samples were selected from kindreds in which three or more family members were affected with pancreatic cancer, at least two of which were first-degree relatives. No mutations were identified in mitogen-activated protein kinase kinase 4 (0 of 22), MADH4 (0 of 22), or ACVR1B (0 of 29), making it unlikely that germ-line mutations in these genes account for a significant number of inherited pancreatic cancers. BRCA2 gene sequencing identified five mutations (5 of 29, 17.2%) that are believed to be deleterious and one point mutation (M192T) unreported previously. Three patients harbored the common 6174delT frameshift mutation, one had the splice site mutation IVS 16-2A > G, and one had the splice site mutation IVS 15-1G > A. Two of the five BRCA2 mutation carriers reported a family history of breast cancer, and none reported a family history of ovarian cancer. These findings confirm the increased risk of pancreatic cancer in individuals with BRCA2 mutations and identify germ-line BRCA2 mutations as the most common inherited genetic alteration yet identified in familial pancreatic cancer.

Activin Receptors, Type I↗

PubMed2002

Simultaneous sequencing of multiple polymerase chain reaction products and combined polymerase chain reaction with cycle sequencing in single reactions.

DNA sequencing is considered the gold standard for nucleic acid identification and mutation detection. However, sequencing is labor intensive because it requires previous amplification and only a single sequence is analyzed at a time. We developed two novel strategies that substantially improve DNA sequencing. The first allows multiple polymerase chain reaction (PCR) products to be sequenced in a single sequencing reaction and analyzed simultaneously in a single lane or capillary. Simultaneous sequencing by this method, designated "SimulSeq," can provide either simultaneous single-direction sequencing of multiple genes or simultaneous forward and reverse sequencing from a single gene. In the second approach, designated "AmpliSeq," we demonstrate a technique combining PCR amplification and sequencing in a single reaction that is analyzed in a single lane or capillary. We demonstrate combined PCR with short bidirectional sequencing, and combined PCR with unidirectional sequencing. We anticipate that these methods will have utility in research and clinical settings where panels of mutations or large numbers of samples are be analyzed and/or when turnaround time is critical.

Base Sequence↗

PubMed2002

High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated Tp53-responsive luciferase reporter.

Human Tp53 is normally a short-lived protein. Tp53 protein is stabilized and levels are increased in response to a variety of cellular stresses, including those induced by genotoxic anticancer drugs and environmental exposures. To engineer an efficient assay based on this property, we constructed and integrated a Tp53-specific reporter system into human cancer cells, termed p53R cells. We tested a range of conventional chemotherapeutic agents as well as over 16 000 diverse small compounds. Ionizing radiation and two-thirds of conventional chemotherapeutic agents, but only 0.2% of diverse compounds activated Tp53 activity by two-fold or greater, consistent with the presumptive genotoxic activation of Tp53 function. Cytotoxicity was independent of TP53 genetic status when paired, syngeneic wild-type TP53 and TP53-null cells in culture were treated with compounds that activated Tp53. From the unbiased survey of random compounds, Tp53 activation was strongly induced by an analog of AMSA, an investigational anti-cancer agent. Tp53 was also strongly induced by an N-oxide of quinoline and by dabequine, an experimental antimalarial evaluated in humans; dabequine was reported to be negative in other screens of mutagenicity and clastogenicity but carcinogenic in animal studies. Further exploration of antimalarial compounds identified the common medicinals chloroquine, quinacrine, and amodiaquine as Tp53-inducers. Flavonoids are known to have DNA topoisomerase activity, a Tp53-inducing activity that is confirmed in the assay. A reported clinical association of Tp53 immunopositive colorectal cancers with use of the antihypertensive agents was extended by the demonstration of hydralazine and nifedipine as Tp53-inducers. p53R cells represent an efficient Tp53 functional assay to identify chemicals and other agents with interesting biologic properties, including genotoxicity. This assay may have utility in the identification of novel chemotherapeutic agents, as an adjunct in the pharmaceutical optimization of lead compounds, in the exploration of environmental exposures, and in chemical probing of the Tp53 pathway.

Amsacrine↗

PubMedDate not supplied

Effects of ambient stimuli on measures of behavioral state and microswitch use in adults with profound multiple impairments.

The effects of different types and amounts of environmental stimuli (visual and auditory) on microswitch use and behavioral states of three individuals with profound multiple impairments were examined. The individual's switch use and behavioral states were measured under three setting conditions: natural stimuli (typical visual and auditory stimuli in a recreational situation), reduced visual stimuli, and reduced visual and auditory stimuli. Results demonstrated differential switch use in all participants with the varying environmental setting conditions. No consistent effects were observed in behavioral state related to environmental condition. Predominant behavioral state scores and switch use did not systematically covary with any participant. Results suggest the importance of considering environmental stimuli in relationship to switch use when working with individuals with profound multiple impairments.

Acoustic Stimulation↗