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Journal of Molecular Diagnostics: JMD

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https://www.readbyqxmd.com/read/28732216/development-of-hla-b-57-01-genotyping-real-time-pcr-with-optimized-hydrolysis-probe-design
#1
Hou-Sung Jung, Gregory J Tsongalis, Joel A Lefferts
HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01...
July 18, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28732215/a-method-for-next-generation-sequencing-of-paired-diagnostic-and-remission-samples-to-detect-mitochondrial-dna-mutations-associated-with-leukemia
#2
Ilaria S Pagani, Chung H Kok, Verity A Saunders, Mark B Van der Hoek, Susan L Heatley, Anthony P Schwarer, Christopher N Hahn, Timothy P Hughes, Deborah L White, David M Ross
Somatic mitochondrial DNA (mtDNA) mutations have been identified in many human cancers, including leukemia. To identify somatic mutations, it is necessary to have a control tissue from the same individual for comparison. When patients with leukemia achieve remission, the remission peripheral blood may be a suitable and easily accessible control tissue, but this approach has not previously been applied to the study of mtDNA mutations. We have developed and validated a next-generation sequencing approach for the identification of leukemia-associated mtDNA mutations in 26 chronic myeloid leukemia patients at diagnosis using either nonhematopoietic or remission blood samples as the control...
July 18, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28732214/crispr-cas9-technology-based-xenograft-tumors-as-candidate-reference-materials-for-multiple-eml4-alk-rearrangements-testing
#3
Rongxue Peng, Rui Zhang, Guigao Lin, Xin Yang, Ziyang Li, Kuo Zhang, Jiawei Zhang, Jinming Li
The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non-small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity...
July 18, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28732213/comparison-of-blood-collection-tubes-from-three-different-manufacturers-for-the-collection-of-cell-free-dna-for-liquid-biopsy-mutation-testing
#4
Christina Alidousty, Danielle Brandes, Carina Heydt, Svenja Wagener, Maike Wittersheim, Stephan Christian Schäfer, Barbara Holz, Sabine Merkelbach-Bruse, Reinhard Büttner, Jana Fassunke, Anne Maria Schultheis
The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples...
July 18, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28727985/comparative-study-and-analytical-verification-of-pcr-methods-for-the-diagnosis-of-congenital-chagas-disease
#5
Carolina Inés Cura, Juan Carlos Ramírez, Marcelo Rodríguez, Constanza Lopez-Albízu, Lucía Irazu, Karenina Scollo, Sergio Sosa-Estani
Congenital infection is currently the first cause of new cases of Chagas disease in Argentina and nonendemic areas worldwide. Its diagnosis is of utmost importance to guarantee curative treatment. To improve such diagnosis, a transfer process of PCR tests to the national laboratory network has been initiated. We performed a comparative study of four PCR assays [two end-point PCR and two duplex real-time quantitative PCR (qPCR) procedures] to detect Trypanosoma cruzi DNA in blood samples. Because satellite DNA and kinetoplastid DNA qPCR methods have the best performance and the use of two different molecular targets for confirmatory purposes has been recommended, these methods selected to perform the transfer process and, in consequence, subjected to an analytical verification protocol based on international guidelines...
July 17, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28727984/gba-analysis-in-next-generation-era-pitfalls-challenges-and-possible-solutions
#6
Stefania Zampieri, Silvia Cattarossi, Bruno Bembi, Andrea Dardis
Mutations in the gene encoding the lysosomal enzyme acid β-glucosidase (GBA) are responsible for Gaucher disease and represent the main genetic risk factor for developing Parkinson disease. In past years, next-generation sequencing (NGS) technology has been applied for the molecular analysis of the GBA gene, both as a single gene or as part of gene panels. However, the presence of complex gene-pseudogene rearrangements, resulting from the presence of a highly homologous pseudogene (GBAP1) located downstream of the GBA gene, makes NGS analysis of GBA challenging...
July 17, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28723342/molecular-analysis-of-circulating-free-dna-from-lung-cancer-patients-in-routine-laboratory-practice-a-cross-platform-comparison-of-three-different-molecular-methods-for-mutation-detection
#7
Stephan Bartels, Sascha Persing, Britta Hasemeier, Elisa Schipper, Hans Kreipe, Ulrich Lehmann
Cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its metastases...
July 16, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28623087/highly-multiplex-real-time-pcr-based-screening-for-blood-borne-pathogens-on-an-openarray-platform
#8
Elena Grigorenko, Carolyn Fisher, Sunali Patel, Valerie Winkelman, Phillip Williamson, Caren Chancey, Germán Añez, Maria Rios, Victoria Majam, Sanjai Kumar, Robert Duncan
Molecular diagnostics are increasingly used in the blood bank industry. A device that can combine simultaneous detection of multiple targets with the flexibility of inclusion of emerging pathogens is desirable for testing blood products. A highly multiplexed blood-borne pathogen panel (BBPP) using dual-label probe chemistry (TaqMan assays) was developed for simultaneous detection and discrimination of 17 viral pathogens in human plasma samples and 13 bacterial and protozoan pathogens in human blood samples on the OpenArray platform...
June 12, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28529006/validation-of-a-next-generation-sequencing-pipeline-for-the-molecular-diagnosis-of-multiple-inherited-cancer-predisposing-syndromes
#9
Paula Paulo, Pedro Pinto, Ana Peixoto, Catarina Santos, Carla Pinto, Patrícia Rocha, Isabel Veiga, Gabriela Soares, Catarina Machado, Fabiana Ramos, Manuel R Teixeira
Despite the growing knowledge of the genetic background behind the cancers that occur in a context of hereditary predisposition, personal or family cancer history may not be clear enough to support directional gene testing. Defined targeted next-generation sequencing gene panels allow identification of the causative disease mutations of multigene syndromes and differential diagnosis for syndromes with phenotypically overlapping characteristics. Herein, we established a next-generation sequencing analysis pipeline for the molecular diagnosis of multiple inherited cancer predisposing syndromes using the commercially available target sequencing panel TruSight Cancer...
May 18, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28506685/simultaneous-genotyping-of-%C3%AE-thalassemia-deletional-and-nondeletional-mutations-by-real-time-pcr-based-multicolor-melting-curve-analysis
#10
Qiuying Huang, Xudong Wang, Ning Tang, Tizhen Yan, Ping Chen, Qingge Li
α-Thalassemia, which is caused by defective synthesis of the hemoglobin α-globin chains, is the most commonly inherited recessive hemoglobin abnormality. Genetic detection of a defective α-globin gene is challenging because of a variety of large deletions of the α-globin gene cluster and nondeletional mutations. Separate detections of them are often required using complex and error-prone open-tube methods. We report a novel real-time PCR-based assay that can simultaneously genotype four major deletional and three common nondeletional mutations in two parallel reactions by using multicolor melting curve analysis...
May 12, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28506684/study-of-preanalytic-and-analytic-variables-for-clinical-next-generation-sequencing-of-circulating-cell-free-nucleic-acid
#11
Meenakshi Mehrotra, Rajesh R Singh, Wei Chen, Richard S P Huang, Alaa A Almohammedsalim, Bedia A Barkoh, Crystal M Simien, Marcos Hernandez, Carmen Behrens, Keyur P Patel, Mark J Routbort, Russell R Broaddus, L Jeffrey Medeiros, Ignacio I Wistuba, Scott Kopetz, Rajyalakshmi Luthra
Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms...
May 12, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28502730/overgrowth-syndromes-caused-by-somatic-variants-in-the-phosphatidylinositol-3-kinase-akt-mammalian-target-of-rapamycin-pathway
#12
REVIEW
Gozde Akgumus, Fengqi Chang, Marilyn M Li
Somatic variants have been well described in tumorigenesis; however, they are only recently appreciated in other human disorders, such as mosaic overgrowth syndromes. Although overgrowth is a manifestation in many genetic syndromes, not all overgrowth syndromes are inherited. Mosaic somatic variants have been lately described in several overgrowth disorders, such as Proteus syndrome, CLOVES (congenital, lipomatous, overgrowth, vascular malformations, epidermal nevi, and spinal/skeletal anomalies and/or scoliosis) syndrome, megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome, and megalencephaly-capillary malformation-polymicrogyria syndrome...
May 11, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28502729/improving-mutation-screening-in-patients-with-colorectal-cancer-predisposition-using-next-generation-sequencing
#13
Jean-Marc Rey, Vincent Ducros, Pascal Pujol, Qing Wang, Marie-Pierre Buisine, Hanaa Aissaoui, Thierry Maudelonde, Sylviane Olschwang
Identification of genetic alterations is important for family risk assessment in colorectal cancers. Next-generation sequencing (NGS) technologies provide useful tools for single-nucleotide and copy number variation (CNV) identification in many genes and samples simultaneously. Herein, we present the validation of current Multiplicom MASTR designs of mismatch repair combined to familial adenomatous polyposis genes in a single PCR reamplification test for eight DNA samples simultaneously on a MiSeq apparatus...
May 11, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28502728/technical-validation-of-a-next-generation-sequencing-assay-for-detecting-clinically-relevant-levels-of-breast-cancer-related-single-nucleotide-variants-and-copy-number-variants-using-simulated-cell-free-dna
#14
Xin Yang, Yuxing Chu, Rui Zhang, Yanxi Han, Lucheng Zhang, Yu Fu, Dan Li, Rongxue Peng, Dongdong Li, Jiansheng Ding, Ziyang Li, Meiru Zhao, Kuo Zhang, Tian Lu, Lang Yi, Qisheng Wu, Guigao Lin, Jiehong Xie, Tao Liu, Ling Yang, Xin Yi, Jinming Li
Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner...
May 11, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28502726/utility-of-nist-whole-genome-reference-materials-for-the-technical-validation-of-a-multigene-next-generation-sequencing-test
#15
Bennett O V Shum, Ilya Henner, Daniele Belluoccio, Marcus J Hinchcliffe
The sensitivity and specificity of next-generation sequencing laboratory developed tests (LDTs) are typically determined by an analyte-specific approach. Analyte-specific validations use disease-specific controls to assess an LDT's ability to detect known pathogenic variants. Alternatively, a methods-based approach can be used for LDT technical validations. Methods-focused validations do not use disease-specific controls but use benchmark reference DNA that contains known variants (benign, variants of unknown significance, and pathogenic) to assess variant calling accuracy of an NGS workflow...
May 11, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28502725/molecular-diagnosis-of-mosaic-overgrowth-syndromes-using-a-custom-designed-next-generation-sequencing-panel
#16
Fengqi Chang, Liu Liu, Erica Fang, Guangcheng Zhang, Tiansheng Chen, Kajia Cao, Yanchun Li, Marilyn M Li
Recent studies have discovered a group of overgrowth syndromes, such as congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, Proteus syndrome, and megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, are caused by somatic activating variants in the genes involved in the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway. Because of the low-abundance nature of these pathogenic variants, Sanger sequencing often yields negative results...
May 11, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28645444/npm1-for-mrd-droplet-like-it-s-hot
#17
Gerald B W Wertheim, Adam Bagg
This commentary highlights the article by Mencia-Trinchant et al that describes a novel digital PCR assay for sensitive detection of minimal residual disease in NPM1 mutated acute myeloid leukemia.
July 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28552549/pheoseq-a-targeted-next-generation-sequencing-assay-for-pheochromocytoma-and-paraganglioma-diagnostics
#18
Maria Currás-Freixes, Elena Piñeiro-Yañez, Cristina Montero-Conde, María Apellániz-Ruiz, Bruna Calsina, Veronika Mancikova, Laura Remacha, Susan Richter, Tonino Ercolino, Natalie Rogowski-Lehmann, Timo Deutschbein, María Calatayud, Sonsoles Guadalix, Cristina Álvarez-Escolá, Cristina Lamas, Javier Aller, Julia Sastre-Marcos, Conxi Lázaro, Juan C Galofré, Ana Patiño-García, Amparo Meoro-Avilés, Judith Balmaña-Gelpi, Paz De Miguel-Novoa, Milagros Balbín, Xavier Matías-Guiu, Rocío Letón, Lucía Inglada-Pérez, Rafael Torres-Pérez, Juan M Roldán-Romero, Cristina Rodríguez-Antona, Stephanie M J Fliedner, Giuseppe Opocher, Karel Pacak, Esther Korpershoek, Ronald R de Krijger, Laurent Vroonen, Massimo Mannelli, Martin Fassnacht, Felix Beuschlein, Graeme Eisenhofer, Alberto Cascón, Fátima Al-Shahrour, Mercedes Robledo
Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue...
July 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28525762/minimal-residual-disease-monitoring-of-acute-myeloid-leukemia-by-massively-multiplex-digital-pcr-in-patients-with-npm1-mutations
#19
Nuria Mencia-Trinchant, Yang Hu, Maria Antonina Alas, Fatima Ali, Bas J Wouters, Sangmin Lee, Ellen K Ritchie, Pinkal Desai, Monica L Guzman, Gail J Roboz, Duane C Hassane
The presence of minimal residual disease (MRD) is widely recognized as a powerful predictor of therapeutic outcome in acute myeloid leukemia (AML), but methods of measurement and quantification of MRD in AML are not yet standardized in clinical practice. There is an urgent, unmet need for robust and sensitive assays that can be readily adopted as real-time tools for disease monitoring. NPM1 frameshift mutations are an established MRD marker present in half of patients with cytogenetically normal AML. However, detection is complicated by the existence of hundreds of potential frameshift insertions, clonal heterogeneity, and absence of sequence information when the NPM1 mutation is identified using capillary electrophoresis...
July 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28502727/concordance-between-research-sequencing-and-clinical-pharmacogenetic-genotyping-in-the-emerge-pgx-study
#20
Laura J Rasmussen-Torvik, Berta Almoguera, Kimberly F Doheny, Robert R Freimuth, Adam S Gordon, Hakon Hakonarson, Jared B Hawkins, Ammar Husami, Lynn C Ivacic, Iftikhar J Kullo, Michael D Linderman, Teri A Manolio, Aniwaa Owusu Obeng, Renata Pellegrino, Cynthia A Prows, Marylyn D Ritchie, Maureen E Smith, Sarah C Stallings, Wendy A Wolf, Kejian Zhang, Stuart A Scott
There has been extensive debate about both the necessity of orthogonal confirmation of next-generation sequencing (NGS) results in Clinical Laboratory Improvement Amendments-approved laboratories and return of research NGS results to participants enrolled in research studies. In eMERGE-PGx, subjects underwent research NGS using PGRNseq and orthogonal targeted genotyping in clinical laboratories, which prompted a comparison of genotyping results between platforms. Concordance (percentage agreement) was reported for 4077 samples tested across nine combinations of research and clinical laboratories...
July 2017: Journal of Molecular Diagnostics: JMD
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