A Variant Detection Pipeline for Inherited Cardiomyopathies–Associated Genes Using Ion Torrent PGM Platform
Publication date: Available online 30 April 2015 Source:The Journal of Molecular Diagnostics Author(s): Théo G.M. Oliveira , Miguel Mitne-Neto , Louise T. Cerdeira , Julia D.C. Marsiglia , Edmundo Arteaga-Fernandez , José E. Krieger , Alexandre C. Pereira In inherited cardiomyopathies, genetic testing is recognized as an enriching procedure in the diagnostic closure of a cardiac condition. Many genetic mutations have been described as pathogenically related to cardiomyopathies, turning next-generation sequencing into an extremely reliable scenario. Here we describe the validation process of a pipeline const...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

The c.1364C>A (p.A455E) Mutation in the CFTR Pseudogene Results in an Incorrectly Assigned Carrier Status by a Commonly Used Screening Platform
We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies
In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Evaluation of HER2 Gene Status in Breast Cancer Samples with Indeterminate Fluorescence in Situ Hybridization by Quantitative Real-Time PCR
Publication date: Available online 5 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Vladimira Koudelakova , Jitka Berkovcova , Radek Trojanec , Jana Vrbkova , Lenka Radova , Jiri Ehrmann , Zdenek Kolar , Bohuslav Melichar , Marian Hajduch Administration of drugs targeting HER2 (official name ERBB2) is an important component of therapy for breast cancer patients with HER2 amplification/overexpression as determined by in situ hybridization (ISH) and immunohistochemistry (IHC). In approximately 5% of breast cancers, ISH assays fail. In these cases, HER2 protein expression is evaluated by IHC alone that may ...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing
In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Analytical Performance of a 15-Gene Prognostic Assay for Early-Stage Non–Small-Cell Lung Carcinoma Using RNA-Stabilized Tissue
Publication date: Available online 8 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Shuguang Huang , Nicholas J. Reitze , Amy L. Ewing , Suzanne McCreary , Arlette H. Uihlein , Stacey L. Brower , Dakun Wang , Tianhua Wang , Michael J. Gabrin , Katherine E. Keating , Jude Mulligan , Claire Wilson , Timothy Davison , Stuart McKenzie , Ming-Sound Tsao , Frances A. Shepherd , Victoria Plamadeala A 15-gene prognostic signature for early-stage, completely resected, non–small-cell lung carcinoma, that distinguishes between patients with good and poor prognoses was clinically validated in prior studies. To...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Annotation of Sequence Variants in Cancer Samples Processes and Pitfalls for Routine Assays in the Clinical Laboratory
Publication date: Available online 11 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Lobin A. Lee , Kevin J. Arvai , Dan Jones As DNA sequencing of multigene panels becomes routine for cancer samples in the clinical laboratory, an efficient process for classifying variants has become more critical. Determining which germline variants are significant for cancer disposition and which somatic mutations are integral to cancer development or therapy response remains difficult, even for well-studied genes such as BRCA1 and TP53. We compare and contrast the general principles and lines of evidence commonly used...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma
Publication date: Available online 14 May 2015 Source:The Journal of Molecular Diagnostics Author(s): John S. Hall , Suzanne Usher , Richard J. Byers , Rebekah C. Higgins , Danish Memon , John A. Radford , Kim M. Linton Personalized management of diffuse large B-cell lymphoma (DLBCL) requires codevelopment of a companion diagnostic assay for activated B-cell and germinal center B-cell subtyping. Current classification methods are costly/complex (microarray expression profiling) or lacking in reproducibility/diagnostic precision (immunophenotyping). We investigated the potential of QuantiGene Plex (QGP)—a branched D...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Development of a Quantitative Real-Time RT-PCR Assay for the Detection of MAGE-A3–Positive Tumors
In conclusion, we have developed a MAGE-A3–specific RT-qPCR assay, compatible with a high-throughput setting for the estimation of MAGEA3 gene expression in present and future clinical trials. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Toward Rare Blood Cell Preservation for RNA Sequencing
Publication date: Available online 16 May 2015 Source:The Journal of Molecular Diagnostics Author(s): Sanja Vickovic , Afshin Ahmadian , Rolf Lewensohn , Joakim Lundeberg Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues—a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the larges...
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

The Evolving Role of the Laboratory Professional in the Age of Genome Sequencing A Vision of the Association for Molecular Pathology
Publication date: Available online 2 June 2015 Source:The Journal of Molecular Diagnostics Author(s): Iris Schrijver , Daniel H. Farkas , Jane S. Gibson , Elaine LyonTeaser The Association for Molecular Pathology emphasizes the need for proactive engagement of laboratory directors with clinicians, patients, and professional organizations as genomic sequence analysis gains importance in diagnostic medicine. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - June 4, 2015 Category: Pathology Source Type: research

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer
Publication date: May 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 3 Author(s): Guanshan Zhu , Xin Ye , Zhengwei Dong , Ya Chao Lu , Yun Sun , Yi Liu , Rose McCormack , Yi Gu , Xiaoqing Liu Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensi...
Source: The Journal of Molecular Diagnostics - April 21, 2015 Category: Pathology Source Type: research

Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology
Publication date: May 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 3 Author(s): Donavan T. Cheng , Talia N. Mitchell , Ahmet Zehir , Ronak H. Shah , Ryma Benayed , Aijazuddin Syed , Raghu Chandramohan , Zhen Yu Liu , Helen H. Won , Sasinya N. Scott , A. Rose Brannon , Catherine O'Reilly , Justyna Sadowska , Jacklyn Casanova , Angela Yannes , Jaclyn F. Hechtman , Jinjuan Yao , Wei Song , Dara S. Ross , Alifya Oultache , Snjezana Dogan , Laetitia Borsu , Meera Hameed , Khedoudja Nafa , Maria E. Arcila , Marc Ladanyi , Michael F. Berger The identification of specific genetic alterations as key oncogeni...
Source: The Journal of Molecular Diagnostics - April 21, 2015 Category: Pathology Source Type: research

Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility? A Report of the Association for Molecular Pathology
Publication date: May 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 3 Author(s): Bert Gold , Milena Cankovic , Larissa V. Furtado , Frederick Meier , Christopher D. Gocke Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization ...
Source: The Journal of Molecular Diagnostics - April 21, 2015 Category: Pathology Source Type: research

HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices
Publication date: May 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 3 Author(s): Scott M. Berry , Alex J. LaVanway , Hannah M. Pezzi , David J. Guckenberger , Meghan A. Anderson , Jennifer M. Loeb , David J. Beebe The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitatio...
Source: The Journal of Molecular Diagnostics - April 19, 2015 Category: Pathology Source Type: research

Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Alicia Edin , Susanne Granholm , Satu Koskiniemi , Annika Allard , Anders Sjöstedt , Anders Johansson Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with line...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

MSK-IMPACT A Hybridization Capture-Based Next-Generation Sequencing Clinical Assay for Solid Tumor Molecular Oncology
Publication date: Available online 20 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Donavan T. Cheng , Talia Mitchell , Ahmet Zehir , Ronak H. Shah , Ryma Benayed , Aijazuddin Syed , Raghu Chandramohan , Zhen Yu Liu , Helen H. Won , Sasinya N. Scott , A. Rose Brannon , Catherine O'Reilly , Justyna Sadowska , Jacklyn Casanova , Angela Yannes , Jaclyn Hechtman , Jinjuan Yao , Wei Song , Dara Ross , Alifya Oultache , Snjezana Dogan , Laetitia Borsu , Meera Hameed , Khedoudja Nafa , Maria E. Arcila , Marc Ladanyi , Michael F. Berger The identification of specific genetic alterations as key oncogenic drive...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

Panels of Cytokines and Other Secretory Proteins as Potential Biomarkers of Ovarian Endometriosis
Publication date: Available online 20 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Vida Kocbek , Katja Vouk , Nick A. Bersinger , Michael D. Mueller , Tea Lanišnik Rižner Endometriosis is a gynecologic disease that is characterized by nonspecific symptoms and invasive diagnostics. To date, there is no adequate noninvasive method for the diagnosis of endometriosis. Although more than 100 potential biomarkers have been investigated in blood and/or peritoneal fluid, none of these has proven useful in clinical practice. The aim to find a suitable panel of biomarkers that would allow noninvasive d...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

Accurate Classification of Germinal Center B-Cell–Like/Activated B-Cell–Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase–Multiplex Ligation-Dependent Probe Amplification Assay A CALYM Study
Publication date: Available online 16 April 2015 Source:The Journal of Molecular Diagnostics Author(s): Sylvain Mareschal , Philippe Ruminy , Cristina Bagacean , Vinciane Marchand , Marie Cornic , Jean-Philippe Jais , Martin Figeac , Jean-Michel Picquenot , Thierry Jo Molina , Thierry Fest , Gilles Salles , Corinne Haioun , Karen Leroy , Hervé Tilly , Fabrice Jardin Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell–like and activated B-cell–like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impe...
Source: The Journal of Molecular Diagnostics - April 18, 2015 Category: Pathology Source Type: research

Evaluation of HPV Genotyping Assays for Archival Clinical Samples
Publication date: Available online 16 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Gabriella Lillsunde Larsson , Jessica Carlsson , Mats G. Karlsson , Gisela Helenius Human papillomavirus (HPV) testing and genotyping of FFPE tissue samples is important in epidemiological investigations. Here, we compare four different HPV genotyping methods for use in FFPE clinical samples. Comparative testing was performed on 99 samples with a clinical suspicion of HPV. Specimens were analyzed with Anyplex II HPV28 detecting 28 genotypes using real-time PCR and melting curve analysis, CLART HPV2 detecting 35 genotyp...
Source: The Journal of Molecular Diagnostics - March 16, 2015 Category: Pathology Source Type: research

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1
Publication date: Available online 14 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Grace X.Y. Lim , Yu Ling Loo , Farmaditya E.P. Mundhofir , Ferdy K. Cayami , Sultana M.H. Faradz , Indhu-Shree Rajan-Babu , Samuel S. Chong , Yvonne Y. Koh , Ming Guan Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but ...
Source: The Journal of Molecular Diagnostics - March 14, 2015 Category: Pathology Source Type: research

Development and Laboratory Evaluation of a Quantitative Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia
Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Alicia Edin , Susanne Granholm , Satu Koskiniemi , Annika Allard , Anders Sjöstedt , Anders Johansson Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with line...
Source: The Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Source Type: research

Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection
Publication date: Available online 13 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Elena Castellanos-Rizaldos , Cloud Paweletz , Chen Song , Geoffrey R. Oxnard , Harvey Mamon , Pasi A. Jänne , G. Mike Makrigiorgos The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple se...
Source: The Journal of Molecular Diagnostics - March 13, 2015 Category: Pathology Source Type: research

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell Lung Cancer
Publication date: Available online 11 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Guanshan Zhu , Xin Ye , Zhengwei Dong , Yachao Lu , Yun Sun , Yi Liu , Rose McCormack , Yi Gu , Xiaoqing Liu Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sens...
Source: The Journal of Molecular Diagnostics - March 11, 2015 Category: Pathology Source Type: research

Improving Molecular Genetic Test Utilization through Order Restriction, Test Review, and Guidance
Publication date: Available online 27 February 2015 Source:The Journal of Molecular Diagnostics Author(s): Jacquelyn D. Riley , Gary W. Procop , Kandice Kottke-Marchant , Robert Wyllie , Felicitas L. Lacbawan The ordering of molecular genetic tests by health providers not well trained in genetics may have a variety of untoward effects. These include the selection of inappropriate tests, the ordering of panels when the assessment of individual or fewer genes would be more appropriate, inaccurate result interpretation and inappropriate patient guidance, and significant unwarranted cost expenditure. We sought to improve the...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

A DNA Real-Time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia
Publication date: March 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 2 Author(s): Paul A. Bartley , Susan Latham , Bradley Budgen , David M. Ross , Elizabeth Hughes , Susan Branford , Deborah White , Timothy P. Hughes , Alexander A. Morley The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by ...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Two Novel Methods for Rapid Detection and Quantification of DNMT3A R882 Mutations in Acute Myeloid Leukemia
We describe a new rapid diagnostic RT-PCR assay based on TauI restriction enzyme reaction to identify DNMT3A R882 mutations at diagnosis. In addition, we developed a sensitive and specific test based on peptide nucleic acid real-time PCR technology to monitor DNMT3A R882H mutation. We identified 24 DNMT3A R882H mutated patients out of 134 acute myeloid leukemia screened samples and we analyzed in these patients the kinetics of minimal residual disease after induction and consolidation therapy. This assay may be useful to better assess response to therapy in patients with acute myeloid leukemia bearing the DNMT3A R882H muta...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Efficient and Highly Sensitive Screen for Myotonic Dystrophy Type 1 Using a One-Step Triplet-Primed PCR and Melting Curve Assay
Publication date: March 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 2 Author(s): Mulias Lian , Indhu-Shree Rajan-Babu , Kunal Singh , Caroline G. Lee , Hai-Yang Law , Samuel S. Chong Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in&n...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

A Novel Technology for Multiplex Gene Expression Analysis Directly from Whole Blood Samples Stabilized at Ambient Temperature Using an RNA-Stabilizing Buffer
We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood sa...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Reporting Incidental Findings in Genomic Scale Clinical Sequencing—A Clinical Laboratory Perspective A Report of the Association for Molecular Pathology
This report by the Association of Molecular Pathology workgroup discusses the pros and cons of next-generation sequencing technology, potential benefits, and harms for reporting of incidental findings, including the effect on both the laboratory and the patient, and compares those with other areas of medicine. The importance of genetic counseling to preserve patient autonomy is also reviewed. The discussion and recommendations presented by the workgroup underline the need for continued research and discussion among all stakeholders to improve our understanding of the effect of different policies on patients, providers, and...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues
Publication date: Available online 4 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Shenli Zhang , Iain BeeHuat Tan , Nur S. Sapari , Heike Grabsch , Alicia Okines , Elizabeth C. Smyth , Toru Aoyama , Lindsay C. Hewitt , Imran Inam , Dan Bottomley , Matthew Nankivell , Sally P. Stenning , David Cunningham , Andrew Wotherspoon , Akira Tsuburaya , Takaki Yoshikawa , Richie Soong , Patrick Tan DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymo...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

A Systematic Approach to Novel Virus Discovery in Emerging Infectious Disease Outbreaks
Publication date: Available online 4 March 2015 Source:The Journal of Molecular Diagnostics Author(s): Siddharth Sridhar , Kelvin K.W. To , Jasper F.W. Chan , Susanna K.P. Lau , Patrick C.Y. Woo , Kwok-Yung Yuen The discovery of novel viruses is of great importance to human health—both in the setting of emerging infectious disease outbreaks and in disease syndromes of unknown etiology. Despite the recent proliferation of many efficient virus discovery methods, careful selection of a combination of methods is important to demonstrate a novel virus, its clinical associations, and its relevance in a timely manner. The...
Source: The Journal of Molecular Diagnostics - March 8, 2015 Category: Pathology Source Type: research

Development of a Genomic DNA Reference Material Panel for Rett Syndrome (MECP2-Related Disorders) Genetic Testing
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Lisa V. Kalman , Jack C. Tarleton , Alan K. Percy , Swaroop Aradhya , Sherri Bale , Shannon D. Barker , Pinar Bayrak-Toydemir , Christina Bridges , Arlene M. Buller-Burckle , Soma Das , Ramaswamy K. Iyer , Timothy D. Vo , Val V. Zvereff , Lorraine H. Toji Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett s...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Gene Expression Ratio Test Distinguishes Normal Lung from Lung Tumors in Solid Tissue and FNA Biopsies
This study supports the hypothesis that the gene-ratio approach reliably distinguishes normal lung from cancerous tissues in FNA samples and can be optimized to diagnose benign nodules. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Sensitive Detection and Serovar Differentiation of Typhoidal and Nontyphoidal Salmonella enterica Species Using 16S rRNA Gene PCR Coupled with High-Resolution Melt Analysis
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Billie J. Masek , Justin Hardick , Helen Won , Samuel Yang , Yu-Hsiang Hsieh , Richard E. Rothman , Charlotte A. Gaydos Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that c...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

A Novel Subtyping Assay for Detection of Clostridium difficile Virulence Genes
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Stephanie L. Angione , Aartik A. Sarma , Aleksey Novikov , Leah Seward , Jennifer H. Fieber , Leonard A. Mermel , Anubhav Tripathi This proof-of-concept study demonstrates the application of a novel nucleic acid detection platform to detect Clostridium difficile in subjects presenting with acute diarrheal symptoms. This method amplifies three genes associated with C. difficile infection, including genes and deletions (cdtB and tcdC) associated with hypervirulence attributed to the NAP1/027/BI strain. Amplification of D...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Molecular Diagnosis of Autosomal Dominant Polycystic Kidney Disease Using Next-Generation Sequencing
In conclusion, the NGS method was superior to Sanger sequencing for detecting PKD gene mutations, achieving high sensitivity and improved gene coverage. These characteristics suggest that NGS would be an appropriate new standard for clinical genetic testing of ADPKD. (Source: The Journal of Molecular Diagnostics)
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia
Publication date: March 2014 Source:The Journal of Molecular Diagnostics, Volume 16, Issue 2 Author(s): Gerald B.W. Wertheim , Catherine Smith , Maria E. Figueroa , Michael Kalos , Adam Bagg , Martin Carroll , Stephen R. Master Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid...
Source: The Journal of Molecular Diagnostics - February 13, 2015 Category: Pathology Source Type: research

Relationship between CFTR and CTRC Variants and the Clinical Phenotype in Late-Onset Cystic Fibrosis Disease with Chronic Pancreatitis
Publication date: Available online 28 January 2015 Source:The Journal of Molecular Diagnostics Author(s): Anna C. Tomaiuolo , Valentina M. Sofia , Cecilia Surace , Fabio Majo , Silvia Genovese , Stefano Petrocchi , Simona Grotta , Federico Alghisi , Vincenzina Lucidi , Adriano Angioni Cystic fibrosis (CF), the most common autosomal recessive disease in whites, is caused by mutations in the CF transmembrane conductance regulator (CFTR). So far, >1900 mutations have been described, most of which are nonsense, missense, and frameshift, and can lead to severe phenotypes, reducing the level of function of the CFTR prot...
Source: The Journal of Molecular Diagnostics - January 28, 2015 Category: Pathology Source Type: research

Target-Enriched Next-Generation Sequencing Reveals Differences between Primary and Secondary Ovarian Tumors in Formalin-Fixed, Paraffin-Embedded Tissue
Publication date: Available online 26 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Stijn Crobach , Dina Ruano , Ronald van Eijk , Gert Jan Fleuren , Ivonne Minderhout , Ronelle Snowdowne , Carli Tops , Tom van Wezel , Hans Morreau Differentiating primary endometrioid or mucinous ovarian tumors from secondary ovarian tumors can be challenging. We compared somatic mutation profiles of primary and secondary ovarian cancers to investigate if these profiles can help diagnose ovarian tumors. Cancer-related genes (n = 115) were screened by target-enriched next-generation sequencing in formalin-fixed, par...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

A DNA Real-time Quantitative PCR Method Suitable for Routine Monitoring of Low Levels of Minimal Residual Disease in Chronic Myeloid Leukemia
Publication date: Available online 29 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Paul A. Bartley , Susan Latham , Bradley Budgen , David M. Ross , Elizabeth Hughes , Susan Branford , Deborah White , Timothy P. Hughes , Alexander A. Morley The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation ...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

High-Throughput Sequencing Using the Ion Torrent Personal Genome Machine for Clinical Evaluation of Somatic Hypermutation Status in Chronic Lymphocytic Leukemia
Publication date: Available online 29 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Rebecca McClure , Ming Mai , Scott McClure For patients with chronic lymphocytic leukemia, an important prognostic indicator is the somatic hypermutation status of immunoglobulin heavy chain variable region nucleic acid within the clonal cell population. Current clinical assays for this evaluation rely on Sanger sequencing and are complex, such that availability of testing for patients is limited and costly. However, advances in high-throughput sequencing provide an opportunity to improve complex clinical sequencing...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

Report of New Haplotype for ABCC2 Gene rs17222723 and rs8187718 in cis
Publication date: Available online 29 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Victoria M. Pratt , Brittany N. Beyer , Daniel L. Koller , Todd C. Skaar , David A. Flockhart , Kenneth D. Levy , Gail H. Vance The ATP-binding cassette, subfamily C [CFTR/MRP], member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across cellular membranes. Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine. Because of the pharmacogenomics implications, we developed a clinical laborat...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

Two Novel Methods for Rapid Detection and Quantification of DNMT3A R882 Mutations in Acute Myeloid Leukemia
We describe a new rapid diagnostic RT-PCR assay based on TauI restriction enzyme reaction to identify DNMT3A R882 mutations at diagnosis. In addition, we developed a sensitive and specific test based on peptide nucleic acid real-time PCR technology to monitor DNMT3A R882H mutation. We identified 24 DNMT3A R882H mutated patients out of 134 acute myeloid leukemia screened samples and we analyzed in these patients the kinetics of minimal residual disease after induction and consolidation therapy. This assay may be useful to better assess response to therapy in patients with acute myeloid leukemia bearing the DNMT3A R882H muta...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

miR-210 Is a Prognostic Marker in Clear Cell Renal Cell Carcinoma
Publication date: Available online 31 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Sara Samaan , Heba W.Z. Khella , Andrew Girgis , Andreas Scorilas , Evi Lianidou , Manal Gabril , Sergey N. Krylov , Michael Jewett , Georg A. Bjarnason , Hala El-said , George M. Yousef Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

Next-Generation Sequencing of the BRCA1 and BRCA2 Genes for the Genetic Diagnostics of Hereditary Breast and/or Ovarian Cancer
Publication date: Available online 31 December 2014 Source:The Journal of Molecular Diagnostics Author(s): Daniel Trujillano , Maximilian E.R. Weiss , Juliane Schneider , Julia Köster , Efstathios B. Papachristos , Viatcheslav Saviouk , Tetyana Zakharkina , Nahid Nahavandi , Lejla Kovacevic , Arndt Rolfs Genetic testing for hereditary breast and/or ovarian cancer mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the BRCA1 and BRCA2 genes. We explored a more efficient genetic screening strategy based on next-generation sequencing of the BRCA1 and BRCA2 genes in 210 heredit...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

Diagnostic Performance of a Cytokine and IFN-γ–Induced Chemokine mRNA Assay after Mycobacterium tuberculosis–Specific Antigen Stimulation in Whole Blood from Infected Individuals
Publication date: January 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 1 Author(s): Sunghyun Kim , Hyejon Lee , Hyunjung Kim , Yeun Kim , Jang-Eun Cho , Hyunwoo Jin , Dae Yeon Kim , Sang-Jun Ha , Young Ae Kang , Sang-Nae Cho , Hyeyoung Lee Interferon (IFN)-γ release assays have limited sensitivity and cannot differentiate between active tuberculosis (TB) disease and latent TB infection (LTBI). Numerous cytokines and regulator factors have been implicated in the pathogenesis and control of Mycobacterium tuberculosis infection. Additional cytokines and chemokines associated with M. tuberculosis ...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

A Clinical Grade Sequencing-Based Assay for CEBPA Mutation Testing Report of a Large Series of Myeloid Neoplasms
Publication date: January 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 1 Author(s): Amir Behdad , Helmut C. Weigelin , Kojo S.J. Elenitoba-Johnson , Bryan L. Betz Diagnostic testing for CEBPA mutations is the standard of care for cytogenetically normal acute myeloid leukemia. Widespread implementation of this testing is hampered by technical challenges associated with the GC content of the gene, the variability of the mutations, and the complexity of the sequence analysis and variant interpretation. We developed a robust Sanger-sequencing test to detect CEBPA mutations in diagnostic acute myeloid le...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

Comparison of Custom Capture for Targeted Next-Generation DNA Sequencing
Publication date: January 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 1 Author(s): Eric Samorodnitsky , Jharna Datta , Benjamin M. Jewell , Raffi Hagopian , Jharna Miya , Michele R. Wing , Senthilkumar Damodaran , Juliana M. Lippus , Julie W. Reeser , Darshna Bhatt , Cynthia D. Timmers , Sameek Roychowdhury Targeted, capture-based DNA sequencing is a cost-effective method to focus sequencing on a coding region or other customized region of the genome. There are multiple targeted sequencing methods available, but none has been systematically investigated and compared. We evaluated four commercially ...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research

High-Quality Genotyping Data from Formalin-Fixed, Paraffin-Embedded Tissue on the Drug Metabolizing Enzymes and Transporters Plus Array
Publication date: January 2015 Source:The Journal of Molecular Diagnostics, Volume 17, Issue 1 Author(s): Hanneke I. Vos , Tahar van der Straaten , Marieke J.H. Coenen , Uta Flucke , D. Maroeska W.M. te Loo , Henk-Jan Guchelaar The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus array covers 1936 markers in 231 genes involved in drug metabolism and transport. Blood- and saliva-derived DNA works well on the DMET array, but the utility of DNA from FFPE tissue has not been reported for this array. As the ability to use DNA from FFPE tissue on the array could open the potential for large retrospective sampl...
Source: The Journal of Molecular Diagnostics - January 4, 2015 Category: Pathology Source Type: research