Analysis of Point Mutations in Clinical Samples of Urothelial Carcinoma

2017 ◽  
pp. 19-28
Author(s):  
Mustafa Alamyar ◽  
Ellen C. Zwarthoff
Keyword(s):  
Urothelial Carcinoma ◽  
Point Mutations ◽  
Clinical Samples

scholarly journals Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations

2001 ◽  
Vol 47 (8) ◽  
pp. 1384-1389 ◽  
Author(s):  
Mark S Turner ◽  
Sarah Penning ◽  
Angela Sharp ◽  
Valentine J Hyland ◽  
Ray Harris ◽  
...  
Keyword(s):  
Solid Phase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Pcr Primers ◽  
Clinical Samples ◽  
Hfe Gene ◽  
Target Sequence ◽  
Nucleotide Polymorphisms ◽  
Nitrophenyl Phosphate ◽  
Hemochromatosis Gene

Abstract Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

scholarly journals Automated fluorescent analysis procedure for enzymatic mutation detection

1998 ◽  
Vol 44 (4) ◽  
pp. 731-739 ◽  
Author(s):  
Benjamin J Del Tito ◽  
Herbert E Poff ◽  
Mark A Novotny ◽  
Donna M Cartledge ◽  
Ralph I Walker ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Signal To Noise Ratio ◽  
Point Mutations ◽  
Suppressor Gene ◽  
Clinical Samples ◽  
High Signal ◽  
Signal To Noise ◽  
Independent Analysis ◽  
Hybridization Time ◽  
Labeled Probe

Abstract The Enzymatic Mutation Detection™ (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism™ 377 DNA Sequencer, that improves on earlier methods (1,2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.

scholarly journals Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1164
Author(s):  
Shona C. Moore ◽  
Rebekah Penrice-Randal ◽  
Muhannad Alruwaili ◽  
Nadine Randle ◽  
Stuart Armstrong ◽  
...  
Keyword(s):  
Viral Genome ◽  
False Negative ◽  
Point Mutations ◽  
Read Length ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Pathogenic Potential ◽  
Number Of Patients ◽  
Interferon Antagonism

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

Clonality of bladder tumors following radical nephroureterectomy: Against the field defect hypothesis.

2015 ◽  
Vol 33 (7_suppl) ◽  
pp. 301-301
Author(s):  
Eugene K. Cha ◽  
John Sfakianos ◽  
Sasinya N. Scott ◽  
Paari Murugan ◽  
Gopa Iyer ◽  
...  
Keyword(s):  
Urothelial Carcinoma ◽  
Point Mutations ◽  
Surgical Techniques ◽  
Median Number ◽  
Intravesical Therapy ◽  
Illustrative Case ◽  
Radical Nephroureterectomy ◽  
Bladder Tumors ◽  
Mutation Profile ◽  
Field Defect

301 Background: Urothelial carcinoma is characterized by multifocal and metachronous tumors. To explain this phenomenon, two hypotheses have been proposed: the ‘field defect’ hypothesis - urothelial cells are primed to undergo transformation by exposure to carcinogens, and the clonal, or ‘single progenitor cell,’ hypothesis - tumors arise from intraluminal seeding of transformed cells. Methods: To examine their clonal relationships, we compared the genomic profiles of primary upper tract urothelial carcinoma (UTUC) tumors and metachronous bladder tumors (intravesical recurrences) in patients treated with radical nephroureterectomy (RNU) and subsequent transurethral resection. Specimens were analyzed using a next-generation, targeted sequencing assay designed to identify point mutations, indels, and copy number alterations in 341 cancer-associated genes. Results: We analyzed 16 primary UTUC tumors and 41 intravesical recurrences in patients treated with RNU. The median number of intravesical recurrences per patient was 2 (range 1-7) and the interval from RNU to intravesical recurrence ranged from 3.5 months to 129 months. With an average sequencing coverage of 516x, we found strong evidence delineating the clonal relationship between primary UTUC tumors and subsequent bladder tumors. The majority of somatic mutations present in the primary UTUC tumors (median=7, range 4-39) were detected in all subsequent bladder tumors (128/146, 88%). In an illustrative case, one patient followed with periodic cystoscopy/cytology who had been NED for 5.5 years then developed 7 bladder tumors over the next 44 months, each with the identical mutation profile (8 mutations) as the primary tumor. Conclusions: We demonstrate that bladder tumors following RNU represent true intravesical recurrences, with almost all tumors sharing the same somatic mutation profile as the primary UTUC tumor. This has important implications for surgical techniques to minimize the risk of intraluminal seeding, the delivery of intravesical therapy following RNU, and the development of strategies employing systemic chemotherapy or targeted agents.

scholarly journals PCR-Based Rapid Identification System Using Bridged Nucleic Acids for Detection of Clarithromycin-Resistant Mycobacterium avium-M. intracellulare Complex Isolates

2016 ◽  
Vol 54 (3) ◽  
pp. 699-704 ◽  
Author(s):  
Takashi Hirama ◽  
Ayako Shiono ◽  
Hiroshi Egashira ◽  
Etsuko Kishi ◽  
Koichi Hagiwara ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Detection System ◽  
Point Mutations ◽  
23S Rrna ◽  
Clinical Samples ◽  
Identification System ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
23S Rrna Gene ◽  
Resistant Mutants

The nontuberculous mycobacteria (NTM) cause miscellaneous disorders in humans, especially in the lungs, which present with a variety of radiological features. To date, knowledge of the pathogenic role of theMycobacterium avium-intracellularecomplex (MAC) in the human lung and the definitive criteria for initiating multidrug therapy are still lacking. However, there is little doubt that clarithromycin is the most efficacious drug among the various treatment regimens for lung NTM. In this study, with the use of a bridged nucleic acid (BNA) probe a detection system based on a real-time PCR (BNA-PCR) for the identification of the point mutations at position 2058 or 2059 in domain V of the 23S rRNA gene responsible for clarithromycin resistance was developed and has been assessed using MAC isolates from clinical samples. Out of 199 respiratory specimens, the drug susceptibility test demonstrated 12 strains resistant to clarithromycin, while the BNA-PCR showed 8 strains carrying the point mutation at position 2058 or 2059 of the 23S rRNA gene. This system revealed that there were mycobacterial strains resistant to clarithromycin which do not carry previously identified resistance genes. This paper documents a novel system for detecting clarithromycin-resistant strains and demonstrates that although these mutations are tacitly assumed to account for >90% of the reported resistant mutants, there is a significant fraction of resistant mutants that do not harbor these mutations. Therefore, unknown mechanisms affecting clarithromycin resistance remain to be elucidated.

scholarly journals A Panel of MicroRNA Signature as a Tool for Predicting Survival of Patients with Urothelial Carcinoma of the Bladder

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Teruo Inamoto ◽  
Hirofumi Uehara ◽  
Yukihiro Akao ◽  
Naokazu Ibuki ◽  
Kazumasa Komura ◽  
...  
Keyword(s):  
High Risk ◽  
Urothelial Carcinoma ◽  
Mirna Expression ◽  
Prognostic Value ◽  
Prognostic Score ◽  
Clinical Samples ◽  
Mirna Signature ◽  
Aggressive Phenotype ◽  
Data Portal ◽  
Carcinoma Of The Bladder

Introduction and Objectives. MicroRNA (miRNA) expression is altered in urologic malignancies, including urothelial carcinoma of the bladder (UCB). Individual miRNAs have been shown to modulate multiple signaling pathways that contribute to BC. To identify a panel of miRNA signature that can predict aggressive phenotype from normal nonaggressive counterpart using miRNA expression levels and to assess the prognostic value of this specific miRNA markers in patients with UCB. Methods. To determine candidate miRNAs as prognostic biomarkers for dividing aggressive type of UCB, miRNA expression was profiled in patients’ samples with an aggressive phenotype or nonaggressive phenotype using 3D-Gene miRNA labeling kit (Toray, Japan). To create a prognostic index model, we used the panel of 9-miRNA signature based on Cancer Genome Atlas (TCGA) data portal (TCGA Data Portal (https://tcgadata.nci.nih.gov/tcga/tcgaHome2.jsp)). miRNA expression data and corresponding clinical data, including outcome and staging information of 84 UCB patients, were obtained. The Kaplan-Meier and log-rank test were performed to quantify the survival functions in two groups. Results. Deregulation of nine miRNAs (hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-145-5p, hsa-miR-4324, hsa-miR-34b-5p, hsa-miR-29c-3p, hsa-miR-135a-3p, and hsa-miR-33b-3p) was determined in UCB patients with aggressive phenotype compared with nonaggressive subject. To validate the prognostic power of the nine-signature miRNAs using the TCGA dataset of bladder cancer, the survival status and tumor miRNA expression of all 84 TCGA UCB patients were ranked according to the prognostic score values. Of nine miRNAs, six were associated with high risk (hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-4324, hsa-miR-34b-5p, and hsa-miR-135a-3p) and three were shown to be protective (hsa-miR-145-5p, hsa-miR-29c-3p, and hsa-miR-33b-3p). Patients with the high-risk miRNA signature exhibited poorer OS than patients expressing the low-risk miRNA profile (HR = 7.05, p<0.001). Conclusions. The miRNA array identified nine dysregulated miRNAs from clinical samples. This panel of nine-miRNA signature provides predictive and prognostic value of patients with UCB.

scholarly journals Validation of multiplex PCR sequencing assay of SIV

2021 ◽  
Author(s):  
Ryan V Moriarty ◽  
Nico Fesser ◽  
Matthew S Sutton ◽  
Vanessa Venturi ◽  
Miles P Davenport ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Sequence Data ◽  
Point Mutations ◽  
Low Frequency ◽  
False Positives ◽  
Starting Material ◽  
Clinical Samples ◽  
Sequencing Data ◽  
Input Material ◽  
Viral Cdna

Abstract Background: The generation of accurate and reproducible viral sequence data is necessary to understand the diversity present in populations of RNA viruses isolated from clinical samples. While various sequencing methods are available, they often require high quality templates and high viral titer to ensure reliable data.Methods: We modified a multiplex PCR and sequencing approach to characterize populations of simian immunodeficiency virus (SIV) isolated from nonhuman primates. We chose this approach with the aim of reducing the number of required input templates while maintaining fidelity and sensitivity. We conducted replicate sequencing experiments using different numbers of quantified viral RNA (vRNA) or viral cDNA as input material. We performed assays with clonal SIVmac239 to detect false positives, and we mixed SIVmac239 and a variant with 24 point mutations (SIVmac239-24X) to measure variant detection sensitivity.Results: We found that utilizing a starting material of quantified viral cDNA templates had a lower rate of false positives and increased reproducibility when compared to that of quantified vRNA templates. This study identifies the importance of rigorously validating deep sequencing methods and including replicate samples when using a new method to characterize low frequency variants in a population with a small number of templates.Conclusions: Because the need to generate reproducible and accurate sequencing data from diverse viruses from low titer samples, we modified a multiplex PCR and sequencing approach to characterize SIV from populations from non-human primates. We found that increasing starting template numbers increased the reproducibility and decreased the number of false positives identified, and this was further seen when cDNA was used as a starting material. Ultimately, we highlight the importance of vigorously validating methods to prevent overinterpretation of low frequency variants in a sample.

scholarly journals c-Myc Acts as a Competing Endogenous RNA to Sponge miR-34a, in the Upregulation of CD44, in Urothelial Carcinoma

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1457 ◽  
Author(s):  
Pie-Che Chen ◽  
Chih-Chia Yu ◽  
Wen-Yu Huang ◽  
Wan-Hong Huang ◽  
Yu-Ming Chuang ◽  
...  
Keyword(s):  
Urothelial Carcinoma ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Promoter Hypermethylation ◽  
Dominant Negative ◽  
Clinical Samples ◽  
Competing Endogenous Rna ◽  
Cd44 Expression ◽  
Common Cancer

MicroRNAs (miRNAs) have been shown to play a crucial role in the progression of human cancers, including urothelial carcinoma (UC), the sixth-most common cancer in the world. Among them, miR-34a has been implicated in the regulation of cancer stem cells (CSCs); however, its role in UC has yet to be fully elucidated. In this study, bioinformatics and experimental analysis confirmed that miR-34a targets CD44 (a CSC surface marker) and c-Myc (a well-known cell cycle regulator) in UC. We found that, surprisingly, most UC cell lines and patient samples did express miR-34a, although epigenetic silencing by promoter hypermethylation of miR-34a expression was observed only in UMUC3 cells, and a subset of patient samples. Importantly, overexpression of c-Myc, a frequently amplified oncogene in UC, was shown to upregulate CD44 expression through a competing endogenous RNA (ceRNA) mechanism, such that overexpression of the c-Myc 3′UTR upregulated CD44, and vice versa. Importantly, we observed a positive correlation between the expression of c-Myc and CD44 in clinical samples obtained from UC patients. Moreover, overexpression of a dominant-negative p53 mutant downregulated miR-34a, but upregulated c-Myc and CD44, in UC cell lines. Functionally, the ectopic expression of miR-34a was shown to significantly suppress CD44 expression, and subsequently, suppression of cell growth and invasion capability, while also reducing chemoresistance. In conclusion, it appears that aberrant promoter methylation, and c-Myc-mediated ceRNA mechanisms, may attenuate the function of miR-34a, in UC. The tumor suppressive role of miR-34a in controlling CSC phenotypes in UC deserves further investigation.

Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

2005 ◽  
Vol 570 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Anna Stanisławska-Sachadyn ◽  
Zygmunt Paszko ◽  
Anna Kluska ◽  
Elżbieta Skasko ◽  
Maria Sromek ◽  
...  
Keyword(s):  
Point Mutations ◽  
Clinical Samples

scholarly journals Upstream Region of OprD Mutations in Imipenem-Resistant and Imipenem-Sensitive Pseudomonas Aeruginosa Isolates

2020 ◽  
Author(s):  
Masoumeh Kiani ◽  
Akram Astani ◽  
Hamed Afkhami ◽  
Mansoor Khaledi ◽  
Javad Fathi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Medical Science ◽  
Point Mutations ◽  
Upstream Region ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Base Substitution ◽  
Coding Region ◽  
Biochemical Tests ◽  
Disk Diffusion Method

Abstract Background: The current study was aimed to investigate the prevalence of the mutations of the oprD gene among imipenem-resistant and -sensitive Pseudomonas aeruginosa isolated from educational hospitals in Yazd, Iran.Methods: In this study, 90 P. aeruginosa isolates were collected from different clinical samples and transferred to the Department of Microbiology, Shahid Sadoghi University of Medical Science, during 2015 to 2016. All isolates were identified by the conventional biochemical tests and antibiotic resistance was determined using disk diffusion method. E. test was performed to determine the minimum inhibitory concentrations (MIC) of imipenem. The mutations of upstream of the oprD coding region and its promoters and 54 primary nucleotide of this gene were recognized by the amplification of this region and subsequently sequenced.Results: Seventy (77.7%) of isolates had MIC≥16 and were resistant to imipenem. The results showed that the rate of resistance to imipenem is increasing. Mutations of the upstream of the oprD gene and its promoters were seen in 25 (86.2%) of isolates and 4 strains had no mutation. All of the imipenem-resistant isolates had mutations. One isolate had a base substitution A→ C at nt 25 in coding region and this isolate had a point mutation leading to an amino acid change at positions 9 (I→L). Conclusion: The results showed that imipenem resistance is increasing in P. aeruginosa, also indicated that the point mutations were the most common cause of the inactivation of upstream of the oprD coding region among P. aeruginosa isolates, it seems this mechanism is effective in resistance of isolates to imipenem.

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