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Journal of Molecular Diagnostics: JMD

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https://www.readbyqxmd.com/read/29792937/a-reliable-targeted-next-generation-sequencing-strategy-for-diagnosis-of-myopathies-and-muscular-dystrophies-especially-for-the-giant-titin-and-nebulin-genes
#1
Reda Zenagui, Delphine Lacourt, Henri Pegeot, Kevin Yauy, Raul Juntas Morales, Corine Theze, François Rivier, Claude Cances, Guilhem Sole, Dimitri Renard, Ulrike Walther-Louvier, Xavier Ferrer-Monasterio, Caroline Espil, Marie-Christine Arné-Bes, Pascal Cintas, Emmanuelle Uro-Coste, Marie-Laure Martin Negrier, Valérie Rigau, Eric Bieth, Cyril Goizet, Mireille Claustres, Michel Koenig, Mireille Cossée
Myopathies and muscular dystrophies (M-MDs) are genetically heterogeneous diseases, with more than 100 identified genes, including the giant and complex titin (TTN) and nebulin (NEB) genes. Next-generation sequencing technology revolutionized M-MD diagnosis and revealed high frequency of TTN and NEB variants. We developed a next-generation sequencing diagnostic strategy targeted on the coding sequences of 135 M-MD genes. Comparison of two targeted capture technologies (SeqCap EZ Choice library capture kit (Roche-Nimblegen) and Nextera Rapid Capture Custom Enrichment kit (Illumina)) and of two whole exome sequencing kits (SureSelect V5 (Agilent) and TruSeq RapidExome capture (Illumina)) revealed best coverage with the SeqCap EZ Choice protocol...
May 21, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29792936/efficient-detection-of-copy-number-mutations-in-pms2-exons-with-close-homologs-detection-of-copy-number-variants-in-3-pms2-exons
#2
Daniel S Herman, Christina Smith, Chang Liu, Cecily P Vaughn, Selvi Palaniappan, Colin C Pritchard, Brian H Shirts
Detection of 3' PMS2 copy number mutations that cause Lynch syndrome is difficult, because of highly homologous pseudogenes. To improve the accuracy and efficiency of clinical screening for these mutations, we developed a new method to analyze standard capture-based, next-generation sequencing data to identify deletions and duplications in PMS2 exons 9 to 15. The approach captures sequence reads using PMS2 targets, maps sequences randomly amongst regions with equal mapping quality, counts reads aligned to homologous exons and introns, and flags read count ratios outside of empirically derived reference ranges...
May 21, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29787863/validity-of-targeted-next-generation-sequencing-in-routine-care-for-identifying-clinically-relevant-molecular-profiles-in-non-small-cell-lung-cancer-results-of-a-2-year-experience-on-1-343-samples
#3
Antoine Legras, Marc Barritault, Anne Tallet, Elizabeth Fabre, Alice Guyard, Bastien Rance, William Digan, Nicolas Pecuchet, Etienne Giroux-Leprieur, Catherine Julie, Stéphane Jouveshomme, Véronique Duchatelle, Véronique Giraudet, Laure Gibault, Alain Cazier, Jean Pastre, Françoise LE Pimpec-Barthes, Pierre Laurent-Puig, Hélène Blons
Theranostic assays are based on single gene testing but the ability of next-generation sequencing (NGS) to interrogate numerous genetic alterations will progressively replace single gene assays. Although NGS was evaluated to screen for theranostic mutations, its usefulness in clinical practice on large series of samples remains to be demonstrated. NGS performance was assessed following guidelines. TaqMan probes and NGS were compared for their ability to detect EGFR and KRAS mutations and NGS mutation profiles were analyzed on a large series of NSCLC (n=1,343)...
May 19, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29704571/detection-of-egfr-variants-in-plasma-a-multilaboratory-comparison-of-the-cobas-egfr-mutation-test-v2-in-europe
#4
Cleo Keppens, John F Palma, Partha M Das, Sidney Scudder, Wei Wen, Nicola Normanno, J Han Van Krieken, Alessandra Sacco, Francesca Fenizia, David Gonzalez de Castro, Selma Hönigschnabl, Izidor Kern, Fernando Lopez-Rios, Maria D Lozano, Antonio Marchetti, Philippe Halfon, Ed Schuuring, Ulrike Setinek, Boe Sorensen, Phillipe Taniere, Markus Tiemann, Hana Vosmikova, Elisabeth M C Dequeker
Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small-cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of the cobas EGFR Mutation Test v2 to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild-type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per mL...
April 25, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29698836/system-for-informatics-in-the-molecular-pathology-laboratory-simpl-an-open-source-end-to-end-solution-for-next-generation-sequencing-clinical-data-management
#5
Wenjun Kang, Sabah Kadri, Rutika Puranik, Michelle N Wurst, Sushant A Patil, Ibro Mujacic, Sonia Benhamed, Nifang Niu, Chao Jie Zhen, Bekim Ameti, Bradley C Long, Filipo Galbo, David Montes, Crystal Iracheta, Venessa L Gamboa, Daisy Lopez, Michael Yourshaw, Carolyn A Lawrence, Dara L Aisner, Carrie Fitzpatrick, Megan E McNerney, Y Lynn Wang, Jorge Andrade, Samuel L Volchenboum, Larissa V Furtado, Lauren L Ritterhouse, Jeremy P Segal
Next-generation sequencing (NGS) diagnostic assays are increasingly becoming the standard of care in oncology practice. As the scale of an NGS laboratory grows, management of these assays requires organizing large amounts of information, including patient data, laboratory processes, genomic data, as well as variant interpretation and reporting. Although several Laboratory Information Systems (LIS) and/or Laboratory Information Management Systems (LIMS) are commercially available, they may not meet all of the needs of a given laboratory, in addition to being frequently cost prohibitive...
April 23, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29698835/suppression-of-wild-type-amplification-by-selectivity-enhancing-agents-in-pcr-assays-that-utilize-superselective-primers-for-the-detection-of-rare-somatic-mutations
#6
Diana Y Vargas, Salvatore A E Marras, Sanjay Tyagi, Fred R Kramer
In PCR assays designed to detect rare somatic mutations, SuperSelective primers, by virtue of their short 3'-foot sequences, selectively initiate synthesis on mutant DNA target fragments, while suppressing the synthesis of related wild-type fragments, and the resulting threshold cycle reflects the quantity of mutant targets present. However, when there are 10 or fewer mutant target fragments in a sample, the threshold cycle that is observed occurs so late that it can be confused with the threshold cycle that arises from samples containing only abundant related wild-type fragments...
April 23, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29689380/mpa-a-free-accessible-and-efficient-pipeline-for-single-nucleotide-variant-annotation-and-prioritization-for-next-generation-sequencing-routine-molecular-diagnosis
#7
Kevin Yauy, David Baux, Henri Pegeot, Charles Van Goethem, Charly Mathieu, Thomas Guignard, Raul Juntas Morales, Delphine Lacourt, Martin Krahn, Vilma-Lotta Lehtokari, Gisele Bonne, Sylvie Tuffery-Giraud, Michel Koenig, Mireille Cossée
Interpretation of next-generation sequencing data constitutes the main limitation in molecular genetics diagnosis. In diagnosis of myopathies and muscular dystrophies (MMD), another major issue is to efficiently predict pathogenicity of variants identified in large genes, especially TTN, since current in silico prediction tools show limitations to predict and rank the numerous variants of such genes. We propose a unique variant prioritization score called mobidic prioritization algorithm (MPA) based on curated interpretation for previously reported variants, biological assumptions, and splice and missense predictors to prioritize all types of single nucleotide variants...
April 21, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29689379/molecular-minimal-residual-disease-monitoring-in-acute-myeloid-leukemia-challenges-and-future-directions
#8
REVIEW
Adrian Selim, Andrew S Moore
The ability to sensitively monitor minimal residual disease (MRD) has played a key role in improving the management and outcomes for a number of leukemias, particularly acute promyelocytic leukemia and childhood acute lymphoblastic leukemia. In contrast, MRD monitoring in acute myeloid leukemia (AML) has been limited by variable assay methodologies and a relative paucity of patient-specific MRD markers. Inter and intratumor genetic heterogeneity pose significant challenges for the identification of molecular markers suitable for MRD monitoring in AML, particularly for those cases without structural chromosomal rearrangements associated with fusion genes...
April 21, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29625250/external-quality-assessment-identifies-training-needs-to-determine-the-neoplastic-cell-content-for-biomarker-testing
#9
Kelly Dufraing, Gert De Hertogh, Véronique Tack, Cleo Keppens, Elisabeth M C Dequeker, Han J H van Krieken
Neoplastic cell content determination is crucial for biomarker testing. It is known that inter-observer variation exists, but large-scale data are missing about variation in tumor delineation and cell content determination between pathologists. Results were obtained from the external quality assessment program for metastatic colorectal cancer from the European Society of Pathology (N=5,776 observations). The study included three parts: current practices were surveyed, neoplastic cell content estimations and delineations were retrieved from stained slides, and clinical reports were analyzed...
April 3, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29625249/accurate-typing-of-human-leukocyte-antigen-class-i-genes-by-oxford-nanopore-sequencing
#10
Chang Liu, Fangzhou Xiao, Jessica Hoisington-Lopez, Kathrin Lang, Philipp Quenzel, Brian Duffy, Robi David Mitra
Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunological compatibility between organ donors and recipients, but the platform's high error rate makes it challenging to type alleles with accuracy. Here, we developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database...
April 3, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29625248/how-to-transfer-a-quantitative-molecular-diagnostic-test-to-multiple-qpcr-platforms
#11
Claudia Gürtler, Mark Laible, Wolfgang Schwabe, Heike Steinhäuser, Xingmin Li, Shujin Liu, Kornelia Schlombs, Ugur Sahin
Quantitative gene expression assays are increasingly used for diagnosis and research, but are often restricted to specific instrumentation. We propose a robust technical and statistical framework that enables transferring of established RT-qPCR assays across qPCR platforms without compromising analytical and clinical validity. The feasibility of our approach on MammaTyper®, an in vitro diagnostic assay which quantifies breast cancer biomarkers and dichotomizes results according to cut-off points, was tested...
April 3, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29625247/added-value-of-50-gene-panel-sequencing-to-distinguish-multiple-primary-lung-cancers-from-pulmonary-metastases-a-systematic-investigation
#12
Paul Roepman, Alexandra Ten Heuvel, Karen C Scheidel, Tobias Sprong, Danielle A M Heideman, Cees A Seldenrijk, Gerarda J M Herder, J Alain Kummer
Differentiation between multiple primary lung cancers or pulmonary metastases has important implications for staging, prognosis, and treatment strategies. Clinical and immuno-histopathological criteria have been standardized; however, a substantial number of cases remain difficult to classify. Using next-generation sequencing it is now possible to improve classification of multiple lung cancer lesions. This study systematically investigated the value of routine morphological and immunohistochemical characteristics, p53 protein expression, TP53 mutation analysis, and 50-gene panel sequencing on 111 lesions from 50 patients with multiple lung lesions...
April 3, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29625246/droplet-digital-pcr-is-a-robust-tool-for-monitoring-minimal-residual-disease-in-adult-philadelphia-positive-acute-lymphoblastic-leukemia
#13
Nicoletta Coccaro, Luisa Anelli, Antonella Zagaria, Paola Casieri, Giuseppina Tota, Paola Orsini, Luciana Impera, Angela Minervini, Crescenzio F Minervini, Cosimo Cumbo, Elisa Parciante, Paola Carluccio, Claudia Brunetti, Giorgina Specchia, Francesco Albano
The BCR-ABL1 p190 fusion transcript (m-bcr) is the most frequent variant observed in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). Qualitative-PCR and real-time quantitative PCR are the currently used methods to monitor minimal residual disease (MRD) in Ph+ ALL patients; for the latter, full standardization and an international quality validation are lacking. Here, we developed a droplet digital PCR (ddPCR) assay for MRD monitoring in p190+ ALL cases. The analytical performance was assessed by the limit of detection determination, demonstrating a reliability, sensitivity, and precision of the assay of up to 0...
April 3, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29572198/hepatitis-b-virus-covalently-closed-circular-dna-selective-droplet-digital-pcr-a-sensitive-and-noninvasive-method-for-hepatocellular-carcinoma-diagnosis
#14
Fan Shen, Consolato Sergi, Hui-Lung Sun
This commentary highlights the article by Huang et al that reports a highly sensitive assay for detection of closed circular DNA of hepatitis B virus.
March 20, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29680088/clustered-regularly-interspaced-short-palindromic-repeat-crispr-crispr-associated-endonuclease-cas9-mediated-homology-independent-integration-for-generating-quality-control-materials-for-clinical-molecular-genetic-testing
#15
Guigao Lin, Kuo Zhang, Rongxue Peng, Yanxi Han, Jiehong Xie, Jinming Li
Genome-edited human cell lines are important resources for producing quality control materials for clinical molecular genetic testing. Generating cell lines with defined mutations through homology-directed repair-based methods are inefficient and can lead to unwanted insertions and deletions in the target loci. Nonhomologous end joining in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system was harnessed to generate genome-engineered cell lines harboring target mutations...
May 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29656833/a-highly-sensitive-and-robust-method-for-hepatitis-b-virus-covalently-closed-circular-dna-detection-in-single-cells-and-serum
#16
Jing-Tao Huang, Ying Yang, Yi-Min Hu, Xing-Hui Liu, Mei-Yan Liao, Roy Morgan, Er-Feng Yuan, Xia Li, Song-Mei Liu
Despite implications of persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the development of hepatocellular carcinoma (HCC), little is known about serum cccDNA in HBV-infected diseases. We developed a cccDNA-selective droplet digital PCR (ddPCR) to assess cccDNA content and dynamics across different stages of HCC development. One hundred forty-seven serum samples and 35 formalin-fixed, paraffin-embedded tumor tissues were derived from patients with HCC or HBV hepatitis/cirrhosis...
May 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29474986/recommendations-for-clinical-cyp2c19-genotyping-allele-selection-a-report-of-the-association-for-molecular-pathology
#17
REVIEW
Victoria M Pratt, Andria L Del Tredici, Houda Hachad, Yuan Ji, Lisa V Kalman, Stuart A Scott, Karen E Weck
This document was developed by the Pharmacogenomics (PGx) Working Group of the Association for Molecular Pathology Clinical Practice Committee, whose aim is to recommend variants for inclusion in clinical pharmacogenomic testing panels. The goals of the Association for Molecular Pathology PGx Working Group are to define the key attributes of PGx alleles recommended for clinical testing and to define a minimum set of variants that should be included in clinical PGx genotyping assays. These recommendations include a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing PGx assays...
May 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29474985/optimal-reference-gene-selection-for-expression-studies-in-human-reticulocytes
#18
Anu Aggarwal, Manu Jamwal, Ganesh K Viswanathan, Prashant Sharma, ManUpdesh S Sachdeva, Deepak Bansal, Pankaj Malhotra, Reena Das
Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and because of several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and at validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected because of published reports...
May 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29474984/chimerism-analysis-in-the-pediatric-setting-direct-pcr-from-bone-marrow-whole-blood-and-cell-fractions
#19
Susanne Kricke, Lana Mhaldien, Rozendo Fernandes, Charizel Villanueva, Alistair Shaw, Paul Veys, Stuart Adams
Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA-extraction step and performing PCR and fragment analysis directly on bone marrow, whole blood, and individual cell fractions. For chimerism analysis, direct-tissue PCR is possible with the use of a robust, commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pretreatment...
May 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29474983/an-in-depth-evaluation-of-the-validity-and-logistics-surrounding-the-testing-of-ar-v7-mrna-expression-in-circulating-tumor-cells
#20
Anieta M Sieuwerts, Bianca Mostert, Michelle van der Vlugt-Daane, Jaco Kraan, Corine M Beaufort, Mai Van, Wendy J C Prager, Bram De Laere, Nick Beije, Paul Hamberg, Hans M Westgeest, Metin Tascilar, Luc Y Dirix, Wendy Onstenk, Ronald de Wit, Martijn P Lolkema, Ron H J Mathijssen, John W M Martens, Stefan Sleijfer
Recent reports have emphasized the clinical relevance of detecting AR-V7 in circulating tumor cells (CTCs). Our aim was to set up a validated multicenter pipeline to measure AR-V7 by quantitative RT-PCR (RT-qPCR) in RNA isolated from CellSearch-enriched CTCs to provide an AR-V7-positive or AR-V7-negative score in a clinically acceptable time range. CellSearch-enirched CTCs from patients with metastatic castration-resistant prostate cancer were characterized by RT-qPCR. After optimization, it was prospectively tested whether it was possible to report the AR-V7 status within 11 days (PRELUDE study)...
May 2018: Journal of Molecular Diagnostics: JMD
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